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1.
Science ; 369(6501): 320-325, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32675374

RESUMO

Restricted V(D)J recombination during fetal development was postulated to limit antibody repertoire breadth and prevent autoimmunity. However, newborn serum contains abundant autoantibodies, suggesting that B cell tolerance during gestation is not yet fully established. To investigate this apparent paradox, we evaluated the reactivities of more than 450 antibodies cloned from single B cells from human fetal liver, bone marrow, and spleen. We found that incomplete B cell tolerance in early human fetal life favored the accumulation of polyreactive B cells that bound both apoptotic cells and commensal bacteria from healthy adults. Thus, the restricted fetal preimmune repertoire contains potentially beneficial self-reactive innate-like B cell specificities that may facilitate the removal of apoptotic cells during development and shape gut microbiota assembly after birth.


Assuntos
Anticorpos/imunologia , Linfócitos B/imunologia , Feto/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoimunidade , Bactérias/imunologia , Feminino , Humanos , Imunidade Inata , Especificidade de Órgãos , Gravidez , Recombinação V(D)J
2.
PLoS One ; 15(7): e0234899, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645020

RESUMO

The increasing prevalence of individuals with multiple food allergies and the need to distinguish between foods containing homologous, cross-reactive proteins have made the use of single-analyte antibody-based methods (e.g., ELISAs) sometimes insufficient. These issues have resulted in the need to conduct multiple analyses and sometimes employ orthogonal methods like mass spectrometry or DNA-based methods for confirmatory purposes. The xMAP Food Allergen Detection Assay (xMAP FADA) was developed to solve this problem while also providing increased throughput and a modular design suitable for adapting to changes in analytical needs. The use of built-in redundancy provides the xMAP FADA with built-in confirmatory analytical capability by including complementary antibody bead sets and secondary analytical end points (e.g., ratio analysis and multi-antibody profiling). A measure of a method's utility is its performance when employed by analysts of varying expertise in multiple laboratory environments. To gauge this aspect, a multi-laboratory validation (MLV) was conducted with 11 participants of different levels of proficiency. The MLV entailed the analysis of incurred food samples in four problematic food matrices, meat sausage, orange juice, baked muffins, and dark chocolate. Except for a couple of instances, involving two confirmatory components in the analysis of baked muffins, the allergenic foods were detected by all participants at concentrations in the analytical samples comparable to ≤ 10 µg/g in the original food sample. In addition, despite high levels of inter-lab variance in the absolute intensities of the responses, the intra-laboratory reproducibility was sufficient to support analyses based on the calibration standards and direct comparison controls (DCCs) analyzed alongside the samples. In contrast, ratio analyses displayed inter-laboratory %CV (RSDR) values < 20%; presumably because the ratios are based on inherent properties of the antigenic elements. The excellent performance of the xMAP FADA when performed by analysts of varying proficiency indicates a reliability sufficient to meet analytical needs.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Hipersensibilidade Alimentar/diagnóstico , Hipersensibilidade Alimentar/imunologia , Alérgenos/imunologia , Anticorpos/imunologia , Bioensaio , Reações Cruzadas , Análise de Alimentos/métodos , Humanos , Laboratórios , Espectrometria de Massas , Reprodutibilidade dos Testes
3.
Proc Natl Acad Sci U S A ; 117(29): 16949-16960, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32616569

RESUMO

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.


Assuntos
Anticorpos/química , Afinidade de Anticorpos , Arginase/imunologia , Regiões Determinantes de Complementaridade/química , Anticorpos/genética , Anticorpos/imunologia , Sítios de Ligação de Anticorpos , Regiões Determinantes de Complementaridade/imunologia , Humanos
4.
PLoS One ; 15(7): e0235563, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645092

RESUMO

Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions. Because western blotting usually involves heat-denaturation of samples prior to gel loading, clarification of detailed procedures for sample preparation have been omitted or neglected in many publications. We show here the case that even excellent primary antibodies failed to detect a specific protein of interest due to a routine heating practice of protein samples. We performed western blotting for transmembrane iron transporter proteins; SLC11A2 (divalent metal transporter 1, DMT1), SLC40A1 (ferroportin 1, Fpn1), and transferrin receptor-1 (TfR1), along with cytoplasmic iron storage protein ferritin H. Our results in 12 human culture cell lysates indicated that only unheated samples prior to gel loading gave rise to clear resolution of DMT1 protein, while heated samples (95°C, 5min) caused the loss of resolution due to DMT1 protein aggregates. Unheated samples also resulted in better resolution for Fpn1 and TfR1 western blots. Conversely, only heated samples allowed to detect ferritin H, otherwise ferritin polymers failed to get into the gel. Neither different lysis/sample loading buffers nor sonication improved the resolution of DMT1 and Fpn1 western blots. Thus, heating samples most critically affected the outcome of western blotting, suggesting the similar cases for thousands of other transmembrane and heat-sensitive proteins.


Assuntos
Western Blotting/métodos , Proteínas de Transporte de Cátions/análise , Fracionamento Celular/métodos , Células A549 , Anticorpos/imunologia , Células CACO-2 , Proteínas de Transporte de Cátions/imunologia , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células Jurkat , Limite de Detecção , Células MCF-7
5.
Yonsei Med J ; 61(7): 606-613, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32608204

RESUMO

PURPOSE: Data on the distribution and impact of panel reactive antibodies (PRA) and donor specific antibodies (DSA) before lung transplantation in Asia, especially multi-center-based data, are limited. This study evaluated the prevalence of and effects of PRA and DSA levels before lung transplantations on outcomes in Korean patients using nationwide multicenter registry data. MATERIALS AND METHODS: This study included 103 patients who received a lung transplant at five tertiary hospitals in South Korea between March 2015 and December 2017. Mortality, primary graft dysfunction (PGD), and bronchiolitis obliterans syndrome (BOS) were evaluated. RESULTS: Sixteen patients had class I and/or class II PRAs exceeding 50%. Ten patients (9.7%) had DSAs with a mean fluorescence intensity (MFI) higher than 1000, six of whom had antibodies with a high MFI (≥2000). DSAs with high MFIs were more frequently observed in patients with high-grade PGD (≥2) than in those with no or low-grade (≤1) PGD. In the 47 patients who survived for longer than 9 months and were evaluated for BOS after the transplant, BOS was not related to DSA or PRA levels. One-year mortality was more strongly related to PRA class I exceeding 50% than that under 50% (0% vs. 16.7%, p=0.007). CONCLUSION: Preoperative DSAs and PRAs are related to worse outcomes after lung transplantation. DSAs and PRAs should be considered when selecting lung transplant recipients, and recipients who have preoperative DSAs with high MFI values and high PRA levels should be monitored closely after lung transplantation.


Assuntos
Anticorpos/imunologia , Bronquiolite Obliterante/etiologia , Transplante de Pulmão/efeitos adversos , Disfunção Primária do Enxerto/imunologia , Doadores de Tecidos , Adulto , Idoso , Bronquiolite Obliterante/diagnóstico , Bronquiolite Obliterante/mortalidade , Feminino , Humanos , Pulmão , Masculino , Pessoa de Meia-Idade , Prevalência , República da Coreia/epidemiologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Análise de Sobrevida
6.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(3): 961-966, 2020 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-32552965

RESUMO

OBJECTIVE: To retrospectively analyze the identification results of irregular antibodies, to clarify the distribution features and to explore the relation of alloantibodies and autoantibodies with the immunized history of patients and disease kinds. METHODS: 49 820 patients who applied for red blood transfusion during Sep 1st 2017 to Sep 1st 2018 were selected. All the specimens were screened for the antibody by microcolumn gel antiglobulin technique, which then were identified for irregular antibody. RESULTS: Antibodies were found in 861 (1.73%) of all 49 820 transfused samples. The alloimmunization history of the patients with antibodies was significantly different between male and female (χ2=18.54,P<0.01). The alloantibody was the most common, accounting for 59.50% in all of the antibodies. Warm autoantibody, anti-E, anti-M, anti-cE and anti-Ce accounted for 68.5% of the antibodies. The blood group of Rh, MNS and Lewis were responsible for 92.40% of alloantibody, especially anti-E accounted for the largest percentage(38.60%) of alloantibody. Patients with alloantiboies experienced much more the alloimmunization and transfusion history (χ2=20.13,P<0.01;χ2=5.40,P<0.05) . The distribution of auto and alloantibody was very significantly different among the ddifferent isease (χ2=51.8,P<0.01), Hematopathy, solid tumor and osteoarthropathy were often associated with alloantibody, otherwise, autoantibodies often occurred in hematopathy and autoimmune disease. CONCLUSION: The most important factor that results in antibody-screening positive is alloantibody, in which anti-E antibody from Rh blood group system in most common.


Assuntos
Anticorpos/imunologia , Eritrócitos , Antígenos de Grupos Sanguíneos , Transfusão de Sangue , Feminino , Humanos , Isoanticorpos , Masculino , Estudos Retrospectivos
7.
Proc Natl Acad Sci U S A ; 117(24): 13509-13518, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32493749

RESUMO

Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid ß (Aß) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aß oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Anticorpos/imunologia , Agregados Proteicos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Animais , Anticorpos/química , Anticorpos/metabolismo , Especificidade de Anticorpos , Caenorhabditis elegans , Modelos Animais de Doenças , Epitopos , Hipocampo/metabolismo , Camundongos , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio Único
8.
Autoimmun Rev ; 19(8): 102592, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32561462

RESUMO

INTRODUCTION: The aim of this narrative review is to provide an overview of the literature on the possible immunologic pathophysiology of psychiatric manifestations of neuropsychiatric systemic lupus erythematosus (NPSLE). METHODS: A systematic search on PubMed was conducted. English studies with full text availability that investigated the correlation between blood-brain barrier (BBB) dysfunction, intrathecal synthesis of antibodies, antibodies, cytokines, chemokines, metalloproteinases, complement and psychiatric NPSLE manifestations in adults were included. RESULTS: Both transient BBB-dysfunction with consequent access of antibodies to the cerebrospinal fluid (CSF) and intrathecal synthesis of antibodies could occur in psychiatric NPSLE. Anti-phospholipid antibodies, anti-NMDA antibodies and anti-ribosomal protein p antibodies seem to mediate concentration dependent neuronal dysfunction. Interferon-α may induce microglial engulfment of neurons, direct neuronal damage and production of cytokines and chemokines in psychiatric NPSLE. Several cytokines, chemokines and matrix metalloproteinase-9 may contribute to the pathophysiology of psychiatric NPSLE by attracting and activating Th1-cells and B-cells. DISCUSSION: This potential pathophysiology may help understand NPSLE and may have implications for the diagnostic management and therapy of psychiatric NPSLE. However, the presented pathophysiological model is based on correlations between potential immunologic etiologies and psychiatric NPSLE that remain questionable. More research on this topic is necessary to further elucidate the pathophysiology of NPSLE.


Assuntos
Anticorpos , Barreira Hematoencefálica , Citocinas , Lúpus Eritematoso Sistêmico , Transtornos Mentais , Anticorpos/imunologia , Barreira Hematoencefálica/imunologia , Quimiocinas/imunologia , Citocinas/imunologia , Humanos , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/imunologia , Transtornos Mentais/etiologia , Transtornos Mentais/imunologia
9.
Gene ; 756: 144911, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32574756

RESUMO

Enolase, a multifunctional glycolytic enzyme, is known to act as a plasminogen receptor in many species, involved in the pivotal processes such as motility, adhesion, invasion, growth, and differentiation of the parasites. Knowledge on the function of enolase from Dermanyssus gallinae is very limited. Here we report on the molecular cloning, enzymatic activity, tissue distribution and plasminogen binding activity of enolase from D. gallinae (DgENO). The full-length of cDNA was 1305 bp, specifying a peptide of 434 amino acids. Bioinformatics analysis showed that DgENO was highly conserved compared with a range of organisms, indicating the potentially similar functions in D. gallinae. A recombinant DgENO (rDgENO) protein was produced and characterized, it catalyzed the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate, the optimal pH was 7.5. Polyclonal antibodies were generated in mice and western blotting indicated that antiserum specifically recognized the native enolase in the somatic extracts from D. gallinae. Immunohistochemical staining of mite sections revealed that the distribution of DgENO was ubiquitous with high level in salivary gland, mite digestive tissues and fat bodies in D. gallinae. Expression level of DgENO was observed mostly in engorged adult mites. Moreover, ELISA binding assay showed that rDgENO could bind plasminogen, and lysine analog ε-aminocaproic acid significantly inhibited this binding activity, indicating that D. gallinae enolase is a receptor of plasminogen. The present study provided foundation for understanding of the biological functions of DgENO and its application in development of vaccines against D. gallinae.


Assuntos
Antígenos/imunologia , Ácaros/imunologia , Fosfopiruvato Hidratase/química , Vacinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Antígenos/química , Antígenos/genética , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Ácaros/enzimologia , Ácaros/genética , Ácaros/crescimento & desenvolvimento , Fosfopiruvato Hidratase/análise , Fosfopiruvato Hidratase/genética , Plasminogênio/metabolismo , Alinhamento de Sequência
10.
Cancer Imaging ; 20(1): 35, 2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398076

RESUMO

BACKGROUND: Anti-angiogenic treatment of glioblastoma (GBM) complicates radiologic monitoring. We evaluated magnetic resonance elastography (MRE) as an imaging tool for monitoring the efficacy of anti-VEGF treatment of GBM. METHODS: Longitudinal studies were performed in an orthotopic GBM xenograft mouse model. Animals treated with B20 anti-VEGF antibody were compared to untreated controls regarding survival (n = 13), classical MRI-contrasts and biomechanics as quantified via MRE (n = 15). Imaging was performed on a 7 T small animal horizontal bore MRI scanner. MRI and MRE parameters were compared to histopathology. RESULTS: Anti-VEGF-treated animals survived longer than untreated controls (p = 0.0011) with progressively increased tumor volume in controls (p = 0.0001). MRE parameters viscoelasticity |G*| and phase angle Y significantly decreased in controls (p = 0.02 for |G*| and p = 0.0071 for Y). This indicates that untreated tumors became softer and more elastic than viscous with progression. Tumor volume in treated animals increased more slowly than in controls, indicating efficacy of the therapy, reaching significance only at the last time point (p = 0.02). Viscoelasticity and phase angle Y tended to decrease throughout therapy, similar as for control animals. However, in treated animals, the decrease in phase angle Y was significantly attenuated and reached statistical significance at the last time point (p = 0.04). Histopathologically, control tumors were larger and more heterogeneous than treated tumors. Vasculature was normalized in treated tumors compared with controls, which showed abnormal vasculature and necrosis. In treated tumors, a higher amount of myelin was observed within the tumor area (p = 0.03), likely due to increased tumor invasion. Stiffness of the contralateral hemisphere was influenced by tumor mass effect and edema. CONCLUSIONS: Anti-angiogenic GBM treatment prolonged animal survival, slowed tumor growth and softening, but did not prevent progression. MRE detected treatment effects on tumor stiffness; the decrease of viscoelasticity and phase angle in GBM was attenuated in treated animals, which might be explained by normalized vasculature and greater myelin preservation within treated tumors. Thus, further investigation of MRE is warranted to understand the potential for MRE in monitoring treatment in GBM patients by complementing existing MRI techniques.


Assuntos
Inibidores da Angiogênese/efeitos adversos , Neoplasias Encefálicas/diagnóstico por imagem , Técnicas de Imagem por Elasticidade/métodos , Glioblastoma/diagnóstico por imagem , Imagem por Ressonância Magnética/métodos , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos/efeitos adversos , Anticorpos/imunologia , Anticorpos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Feminino , Glioblastoma/tratamento farmacológico , Camundongos , Camundongos Nus , Fator A de Crescimento do Endotélio Vascular/imunologia
11.
PLoS Comput Biol ; 16(5): e1007840, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365062

RESUMO

We present a flexible, open source R package designed to obtain biological and epidemiological insights from serological datasets. Characterising past exposures for multi-strain pathogens poses a specific statistical challenge: observed antibody responses measured in serological assays depend on multiple unobserved prior infections that produce cross-reactive antibody responses. We provide a general modelling framework to jointly infer infection histories and describe immune responses generated by these infections using antibody titres against current and historical strains. We do this by linking latent infection dynamics with a mechanistic model of antibody kinetics that generates expected antibody titres over time. Our aim is to provide a flexible package to identify infection histories that can be applied to a range of pathogens. We present two case studies to illustrate how our model can infer key immunological parameters, such as antibody titre boosting, waning and cross-reaction, as well as latent epidemiological processes such as attack rates and age-stratified infection risk.


Assuntos
Anticorpos/sangue , Anticorpos/imunologia , Humanos , Incidência , Cinética , Modelos Biológicos
12.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(3): 264-270, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32389175

RESUMO

Objective To express E6 protein of human papillomavirus (HPV) type 16 in prokaryotic expression system and prepare its polyclonal antibody. Methods HPV16 E6 gene was obtained from Siha cells by PCR and cloned into pET21a(+) vector to construct the recombinant plasmid pET21a(+)/HPV16 E6 that was confirmed by sequencing. The recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3). The HPV16 E6-His tag recombinant protein was expressed after the induction of isopropyl beta-D-1-thiogalactopyranoside (IPTG), purified by Ni-NTA affinity chromatography, and then analyzed by Western blot analysis. The purified HPV16 E6 recombinant protein was used to immunize Japanese white rabbits to prepare polyclonal antibody. The titer of the serum polyclonal antibody was determined by ELISA. The specificity of the polyclonal antibody was analyzed by Western blotting and immunofluorescence. Results The recombinant plasmid pET21a(+)/HPV16 E6 was successfully constructed and confirmed by sequencing. After the recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3), the recombinant HPV16 E6 protein was expressed and purified by affinity chromatography. The polyclonal antibody at a titer of 1:40 000 was obtained by immunizing Japanese big-ear white rabbit with the purified recombinant HPV16 E6 protein, and its specificity was confirmed by Western blotting and immunofluorescence assay. Conclusion HPV16 E6 recombinant protein was successfully expressed and the rabbit polyclonal antibody against HPV16 E6 recombinant protein was prepared.


Assuntos
Anticorpos/imunologia , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas Virais/imunologia , Proteínas Repressoras/biossíntese , Proteínas Repressoras/imunologia , Animais , Especificidade de Anticorpos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Plasmídeos , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia
13.
Arch Virol ; 165(5): 1163-1176, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32232673

RESUMO

Monoclonal antibodies have attracted wide attention in therapeutics owing to their high efficacy, low toxicity, and specific targeting. However, antibodies cannot cross the cell membrane barrier. Therefore, their therapeutic potential is limited to surface-exposed antigens or secreted proteins. In the present investigation, we have developed a chimeric virus-like particle (VLP) of pepper vein banding virus (PVBV) and explored the possibility of using it as a delivery vehicle for antibodies against intracellular antigens as well as for future applications in immunodiagnostics. The chimeric PVBV particles were generated by genetically engineering the B domain of Staphylococcus aureus protein A (SpA) at the N-terminus of the PVBV coat protein (CP). The chimeric VLPs purified by sucrose density gradient centrifugation had ~440-fold higher affinity towards IgG antibody when compared to SpA. Interestingly, the unassembled chimeric CP with the B-domain at the N-terminus (BCP) purified by Ni-NTA chromatography was a monomer, and it had ~45-fold higher affinity towards antibodies compared to SpA. Additionally, the chimeric particles were able to bind and deliver antibodies against both intracellular (α-tubulin) and surface-exposed antigens (CD 20). However, the BCP monomer failed to enter mammalian cells. Thus, for the first time, we have demonstrated that the assembled VLPs are essential for internalization. These results demonstrate the potential of the use of chimeric PVBV VLPs in diagnostics and, more importantly, as nanocarriers for intracellular delivery of antibodies.


Assuntos
Anticorpos/metabolismo , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Endocitose , Potyvirus/genética , Virossomos/genética , Animais , Anticorpos/imunologia , Proteínas do Capsídeo/genética , Linhagem Celular , Humanos , Proteínas Recombinantes de Fusão/genética , Recombinação Genética , Proteína Estafilocócica A/genética
14.
Plant Mol Biol ; 103(6): 597-608, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32346812

RESUMO

KEY MESSAGE: Nanobody-heavy chain (VHH-Fc) antibody formats have the potential to immunomodulate even highly accumulating proteins and provide a valuable tool to experimentally modulate the subcellular distribution of seed storage proteins. Recombinant antibodies often obtain high accumulation levels in plants, and thus, besides being the actual end-product, antibodies targeting endogenous host proteins can be used to interfere with the localization and functioning of their corresponding antigens. Here, we compared the effect of a seed-expressed nanobody-heavy chain (VHH-Fc) antibody against the highly abundant Arabidopsis thaliana globulin seed storage protein cruciferin with that of a VHH-Fc antibody without endogenous target. Both antibodies reached high accumulation levels of around 10% of total soluble protein, but strikingly, another significant part was present in the insoluble protein fraction and was recovered only after extraction under denaturing conditions. In seeds containing the anti-cruciferin antibodies but not the antibody without endogenous target, the amount of soluble, processed globulin subunits was severely reduced and a major part of the cruciferin molecules was found as precursor in the insoluble fraction. Moreover, in these seeds, aberrant vacuolar phenotypes were observed that were different from the effects caused by the depletion of globulins in knock-out seeds. Remarkably, the seeds with strongly reduced globulin amounts are fully viable and germinate with frequencies similar to wild type, illustrating how flexible seeds can retrieve amino acids from the stored proteins to start germination.


Assuntos
Anticorpos/imunologia , Anticorpos/metabolismo , Globulinas/imunologia , Proteínas de Armazenamento de Sementes/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas de Armazenamento de Sementes/genética , Vacúolos/metabolismo
15.
Anticancer Res ; 40(3): 1467-1473, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32132045

RESUMO

BACKGROUND: BTH1677 is a beta-glucan pathogen-associated molecular pattern (PAMP) being evaluated as a novel immunotherapy of cancer. We previously described that the presence of antibodies against beta-glucan (ABA) in serum is necessary for BTH1677 antitumoral activity. We hypothesized that infusion of immunoglobulin can reinstate responses to BTH1677 in individuals with low ABA levels. PATIENTS AND METHODS: We report two single-patient studies: one in a patient with metastatic colorectal cancer who received BTH1677, combined with tumor targeting antibody cetuximab; and a second in a patient with metastatic neuroendocrine tumor who received BTH1677 combined with immune checkpoint inhibitor pembrolizumab. RESULTS: The patients had low serum titers of ABA and low innate immune effector functionality induced by BTH1677. Addition of intravenous immunoglobulins restored innate immune activity of BTH1677 and induced clinically meaningful anti-tumoral activity, with long-term disease control. CONCLUSION: Infusion of immunoglobulin can restore activity of BTH1677 in individuals with low serum ABA level.


Assuntos
Anticorpos/sangue , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Glucanos/administração & dosagem , Tumores Neuroendócrinos/imunologia , Tumores Neuroendócrinos/terapia , beta-Glucanas/imunologia , Idoso de 80 Anos ou mais , Anticorpos/imunologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos Imunológicos/administração & dosagem , Cetuximab/administração & dosagem , Feminino , Humanos , Imunoglobulinas/administração & dosagem , Imunoterapia/métodos , Pessoa de Meia-Idade
16.
PLoS One ; 15(3): e0230267, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160634

RESUMO

PURPOSE: Rapid, intra-operative identification of tumor tissue in the margins of excised specimens has become an important focus in the pursuit of reducing re-excision rates, especially for breast conserving surgery. Dual-probe difference specimen imaging (DDSI) is an emerging approach that uses the difference in uptake/clearance kinetics between a pair of fluorescently-labeled stains, one targeted to a biomarker-of-interest and the other an untargeted isotype, to reveal receptor-specific images of the specimen. Previous studies using antibodies labeled with either enhanced Raman particles or organic fluorophores have shown promising tumor vs. normal diagnostic performance. Yet, the unique properties of quantum dot-labeled antibody complexes (QDACs), which provide spectrally-distinct fluorescence emission from a common excitation source, make them ideal candidates for this application. Herein, we evaluate the diagnostic performance of QDAC-based DDSI in excised xenografts. PROCEDURES: Excised fresh specimens of normal tissue and human tumor xenografts with elevated expression of HER2 were stained with a HER2-targeted QDAC and an untargeted QDAC isotype. Stained specimens were imaged on a custom hyperspectral imaging system capable of spectrally separating the quantum dot signatures, and images processed using the DDSI approach. The diagnostic performance of this technique under different incubation temperatures and probe concentrations was evaluated using receiver-operator characteristic analysis. RESULTS: HER2-targeted QDAC-DDSI was able to distinguish HER2(+) tumors from normal tissue with reasonably high diagnostic performance; however, this performance was sensitive to temperature during the staining procedure. Area under the curve values were 0.61 when staining at room temperature but increased to over 0.81 when staining at 37 °C. Diagnostic performance was not affected by increasing stain concentration. CONCLUSIONS: This study is the first to report dual-probe difference imaging of specimens using QDACs and hyperspectral imaging. Our results show promising diagnostic performance under certain conditions, and compel further optimization and evaluation of this intra-operative margin assessment technique.


Assuntos
Biomarcadores Tumorais/imunologia , Neoplasias Mamárias Experimentais/diagnóstico , Pontos Quânticos , Animais , Anticorpos/imunologia , Feminino , Humanos , Imunoensaio/métodos , Imunoensaio/normas , Células MCF-7 , Camundongos , Camundongos Nus , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/normas , Receptor ErbB-2/imunologia
17.
PLoS One ; 15(3): e0227165, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32218565

RESUMO

AIM: Abdominal aortic aneurysms (AAA) is a life-threatening weakening and expansion of the abdominal aorta due to inflammatory cell infiltration and gradual degeneration of extracellular matrix (ECM). There are no pharmacological therapies to treat AAA. We tested the hypothesis that nanoparticle (NP) therapy that targets degraded elastin and delivers anti-inflammatory, anti-oxidative, and ECM stabilizing agent, pentagalloyl glucose (PGG) will reverse advance stage aneurysm in an elastase-induced mouse model of AAA. METHOD AND RESULTS: Porcine pancreatic elastase (PPE) was applied periadventitially to the infrarenal aorta in mice and AAA was allowed to develop for 14 days. Nanoparticles loaded with PGG (EL-PGG-NPs) were then delivered via IV route at 14-day and 21-day (10 mg/kg of body weight). A control group of mice received no therapy. The targeting of NPs to the AAA site was confirmed with fluorescent dye marked NPs and gold NPs. Animals were sacrificed at 28-d. We found that targeted PGG therapy reversed the AAA by decreasing matrix metalloproteinases MMP-9 and MMP-2, and the infiltration of macrophages in the medial layer. The increase in diameter of the aorta was reversed to healthy controls. Moreover, PGG treatment restored degraded elastic lamina and increased the circumferential strain of aneurysmal aorta to the healthy levels. CONCLUSION: Our results support that site-specific delivery of PGG with targeted nanoparticles can be used to treat already developed AAA. Such therapy can reverse inflammatory markers and restore arterial homeostasis.


Assuntos
Aorta Abdominal/efeitos dos fármacos , Aneurisma da Aorta Abdominal/tratamento farmacológico , Portadores de Fármacos/química , Taninos Hidrolisáveis/administração & dosagem , Imunoconjugados/administração & dosagem , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Aorta Abdominal/diagnóstico por imagem , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Modelos Animais de Doenças , Elastina/antagonistas & inibidores , Elastina/imunologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/patologia , Ouro , Humanos , Imunoconjugados/imunologia , Injeções Intravenosas , Masculino , Nanopartículas Metálicas/química , Camundongos , Elastase Pancreática/administração & dosagem , Elastase Pancreática/toxicidade , Soroalbumina Bovina/química , Ultrassonografia
18.
Cancer Sci ; 111(5): 1750-1760, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32061104

RESUMO

Hepatocellular carcinoma (HCC) is a common and particularly fatal form of cancer for which very few drugs are effective. The fibroblast growth factor 19 (FGF19) has been viewed as a driver of HCC development and a potential Ab target for developing novel HCC therapy. However, a previously developed anti-FGF19 Ab disrupted FGF19's normal regulatory function and caused severe bile-acid-related side-effects despite of having potent antitumor effects in preclinical models. Here, we developed novel human Abs (G1A8 and HS29) that specifically target the N-terminus of FGF19. Both Abs inhibited FGF19-induced HCC cell proliferation in vitro and significantly suppressed HCC tumor growth in mouse models. Importantly, no bile-acid-related side effects were observed in preclinical cynomolgus monkeys. Fundamentally, our study demonstrates that it is possible to target FGF19 for anti-HCC therapies without adversely affecting its normal bile acid regulatory function, and highlights the exciting promise of G1A8 or HS29 as potential therapy for HCC.


Assuntos
Anticorpos/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Fatores de Crescimento de Fibroblastos/imunologia , Neoplasias Hepáticas/tratamento farmacológico , Animais , Anticorpos/química , Anticorpos/imunologia , Antineoplásicos/química , Antineoplásicos/imunologia , Ácidos e Sais Biliares/sangue , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular , Modelos Animais de Doenças , Epitopos , Feminino , Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Macaca fascicularis , Masculino , Camundongos
19.
Eur J Histochem ; 64(1)2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32046476

RESUMO

The gene expression and protein synthesis of small leucine-rich proteoglycans (SLRPs), including decorin, biglycan, fibromodulin, and lumican, was analyzed in the context of the hypothesis that they are closely related to tooth formation. In situ hybridization, immunohistochemistry, and organ culture with metabolic labeling of [35S] were carried out in mouse first molar tooth germs of different developmental stages using ICR mice at embryonic day (E) 13.5 to postnatal day (P) 7.0. At the bud and cap stage, decorin mRNA was expressed only in the surrounding mesenchyme, but not within the tooth germ. Biglycan mRNA was then expressed in the condensing mesenchyme and the dental papilla of the tooth germ. At the apposition stage (late bell stage), both decorin and biglycan mRNA were expressed in odontoblasts, resulting in a switch of the pattern of expression within the different stages of odontoblast differentiation. Decorin mRNA was expressed earlier in newly differentiating odontoblasts than biglycan. With odontoblast maturation and dentin formation, decorin mRNA expression was diminished and localized to the newly differentiating odontoblasts at the cervical region. Simultaneously, biglycan mRNA took over and extended its expression throughout the new and mature odontoblasts. Both mRNAs were expressed in the dental pulp underlying the respective odontoblasts. At P7.0, both mRNAs were weakly expressed but maintained their spatial expression patterns. Immunostaining showed that biglycan was localized in the dental papillae and pulp. In addition, all four SLRPs showed clear immunostaining in predentin, although the expressions of fibromodulin and lumican mRNAs were not identified in the tooth germs examined. The organ culture data obtained supported the histological findings that biglycan is more predominant than decorin at the apposition stage. These results were used to identify biglycan as the principal molecule among the SLRPs investigated. Our findings indicate that decorin and biglycan show spatial and temporal differential expressions and play their own tissue-specific roles in tooth development.


Assuntos
Dente Molar/embriologia , Odontogênese/fisiologia , Proteoglicanos Pequenos Ricos em Leucina/metabolismo , Germe de Dente/metabolismo , Animais , Anticorpos/imunologia , Feminino , Expressão Gênica/fisiologia , Imuno-Histoquímica , Camundongos Endogâmicos ICR , Dente Molar/química , Dente Molar/citologia , Odontogênese/genética , Gravidez , RNA Mensageiro/metabolismo , Coelhos , Proteoglicanos Pequenos Ricos em Leucina/genética , Proteoglicanos Pequenos Ricos em Leucina/imunologia , Germe de Dente/química , Germe de Dente/citologia , Germe de Dente/crescimento & desenvolvimento
20.
Food Chem ; 317: 126376, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32078991

RESUMO

We and others have identified biomarker candidates of tenderness or marbling, two major attributes of bovine meat-eating qualities for consumers' satisfaction. In this study, Reverse Phase Protein Arrays (RPPA) and targeted mass spectrometry assays using Parallel Reaction Monitoring (PRM) were developed to test whether 10 proteins pass the sequential qualification and verification steps of the challenging biomarker discovery pipeline. At least MYH1, TPI1, ALDH1A1 and CRYAB were qualified by RPPA or PRM as being differentially abundant according to marbling values of longissimus thoracis and semimembranosus muscles. Significant mathematical relationships between the individual abundance of each of the four proteins and marbling values were verified by linear or logistic regressions. Four proteins, TNNT1, MDH1, PRDX6 and ENO3 were qualified and verified for tenderness, and the abundance of MDH1 explained 49% of the tenderness variability. The present PRM and RPPA results pave the way for development of useful meat industrial multiplex-proteins assays.


Assuntos
Anticorpos/imunologia , Biomarcadores/análise , Carne/análise , Proteômica/métodos , Aldeído Desidrogenase 1/análise , Aldeído Desidrogenase 1/imunologia , Animais , Anticorpos/análise , Bovinos , Limite de Detecção , Modelos Lineares , Modelos Logísticos , Espectrometria de Massas , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/análise , Cadeias Pesadas de Miosina/imunologia , Análise Serial de Proteínas
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