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1.
Nature ; 611(7935): 352-357, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36289331

RESUMO

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens1. In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens2-22. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes23,24. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.


Assuntos
Anticorpos , Seleção Clonal Mediada por Antígeno , Cadeias Pesadas de Imunoglobulinas , Cadeias Leves de Imunoglobulina , Animais , Sequência de Aminoácidos , Anticorpos/química , Anticorpos/genética , Anticorpos/imunologia , Antígenos/química , Antígenos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Mamíferos , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Memória Imunológica , Recombinação V(D)J , Seleção Clonal Mediada por Antígeno/genética , Seleção Clonal Mediada por Antígeno/imunologia
2.
World J Surg Oncol ; 20(1): 336, 2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36207751

RESUMO

BACKGROUND: TIMM8A is a protein-coding gene located on the X chromosome. There is evidence that TIMM8A plays an important role in mitochondrial morphology and fission. Studies have shown that mitophagy and fission could affect the function of immune cells. However, there is currently no research on this gene's role in cancer occurrence and progression. METHODS: TIMM8A expression was analyzed via the Tumor Immune Estimation Resource (TIMER) site and UALCAN database. We evaluated the influence of TIMM8A on clinical prognosis using Kaplan-Meier plotter, the PrognoScan database, and Human Protein Atlas (HPA). The correlations between TIMM8A and cancer immune infiltrates were investigated via TIMER. Tumor Immune Dysfunction and Exclusion (TIDE) was used to evaluate the potential of tumor immune evasion. Functions of TIMM8A mutations and 50 genes significantly associated with TIMM8A mutations in breast cancer (BRCA) and uterine corpus endometrial cancer (UCEC) were analyzed by GO and KEGG in LinkedOmics database. RESULTS: We investigated the role of TIMM8A in multiple cancers and found that it was significantly associated with poor prognosis in BRCA and UCEC. After analyzing the effect of TIMM8A on immune infiltration, we found Th2 CD4+ T cells might be a common pathway by which TIMM8A contributed to poor prognosis in BRCA and UCEC. Our results suggested that myeloid-derived suppressor cells (MDSC) and tumor-associated M2 macrophages (TAM M2) might be important factors in immune evasion through T cell rejection in both cancers, and considered TIMM8A as a biomarker to predict the efficacy of this therapy in BRCA and UCEC. The results of TIMM8A enrichment analysis showed us that abnormally expressed TIMM8A might affect the mitochondrial protein in BRCA and UCEC. CONCLUSIONS: Contributed to illustrating the value of TIMM8A as a prognostic biomarker, our findings suggested that TIMM8A was correlated with prognosis and immune infiltration, including CD8+ T cells, Th2 CD4+ T cells, and macrophages in BRCA and UCEC. In addition, TIMM8A might affect immune infiltration and prognosis in BRCA and UCEC by affecting mitophagy. We believed it could also be a biomarker to predict the efficacy of anti-PD-L1 therapy and proposed to improve the efficacy by eliminating MDSC and TAM M2.


Assuntos
Anticorpos/imunologia , Antígeno B7-H1/imunologia , Neoplasias da Mama , Carcinoma Endometrioide , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Feminino , Humanos , Proteínas de Membrana Transportadoras , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais , Prognóstico
3.
Front Immunol ; 13: 931210, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36091034

RESUMO

Atypical hemolytic uremic syndrome (aHUS) an important form of a thrombotic microangiopathy (TMA) that can frequently lead to acute kidney injury (AKI). An important subset of aHUS is the anti-factor H associated aHUS. This variant of aHUS can occur due to deletion of the complement factor H genes, CFHR1 and CFHR3, along with the presence of anti-factor H antibodies. However, it is a point of interest to note that not all patients with anti-factor H associated aHUS have a CFHR1/R3 deletion. Factor-H has a vital role in the regulation of the complement system, specifically the alternate pathway. Therefore, dysregulation of the complement system can lead to inflammatory or autoimmune diseases. Patients with this disease respond well to treatment with plasma exchange therapy along with Eculizumab and immunosuppressant therapy. Anti-factor H antibody associated aHUS has a certain genetic predilection therefore there is focus on further advancements in the diagnosis and management of this disease. In this article we discuss the baseline characteristics of patients with anti-factor H associated aHUS, their triggers, various treatment modalities and future perspectives.


Assuntos
Injúria Renal Aguda , Síndrome Hemolítico-Urêmica Atípica , Proteínas do Sistema Complemento , Injúria Renal Aguda/genética , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/terapia , Anticorpos/genética , Anticorpos/imunologia , Síndrome Hemolítico-Urêmica Atípica/complicações , Síndrome Hemolítico-Urêmica Atípica/genética , Síndrome Hemolítico-Urêmica Atípica/imunologia , Síndrome Hemolítico-Urêmica Atípica/terapia , Proteínas Sanguíneas/genética , Proteínas Inativadoras do Complemento C3b/genética , Fator H do Complemento/antagonistas & inibidores , Fator H do Complemento/genética , Fator H do Complemento/imunologia , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/imunologia , Humanos , Troca Plasmática
4.
Nature ; 610(7930): 182-189, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36131013

RESUMO

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.


Assuntos
Anticorpos , Especificidade de Anticorpos , Proteínas de Membrana , Proteólise , Ubiquitina-Proteína Ligases , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Neoplasias Colorretais/metabolismo , Ligantes , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Especificidade por Substrato , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo
5.
J Neuroimmunol ; 370: 577932, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35853357

RESUMO

A significant proportion of multiple sclerosis (MS) patients treated with interferon beta-1a (Rebif™) develop anti-drug antibodies (ADA) with a negative impact on treatment efficacy. We hypothesized that high-throughput B-cell receptor (BCR) repertoire analysis could be used to predict and monitor ADA development. To study this we analyzed 228 peripheral blood samples from 68 longitudinally followed patients starting on interferon beta-1a. Our results show that whole blood BCR analysis does not reflect, and does not predict ADA development in MS patients treated with interferon beta-1a. We propose that BCR analysis of phenotypically selected cell subsets or tissues might be more informative.


Assuntos
Esclerose Múltipla , Anticorpos/imunologia , Humanos , Interferon beta-1a/efeitos adversos , Interferon beta-1a/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Receptores de Antígenos de Linfócitos B/sangue , Receptores de Antígenos de Linfócitos B/imunologia
6.
PLoS One ; 17(7): e0268848, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35776709

RESUMO

The Rho family Guanine nucleotide exchange factor (GEF) ARHGEF17 (also known as TEM4) is a large protein with only 3 annotated regions: an N-terminal actin-binding domain, a Rho-specific dbl homology (DH)- pleckstrin homology (PH) type GEF domain and a seven bladed ß propeller fold at the C-terminus with unknown function. TEM4 has been implicated in numerous activities that rely on regulation of the cytoskeleton including cell migration, cell-cell junction formation and the spindle assembly checkpoint during mitosis. Here we have assessed the specificity of a TEM4 polyclonal antibody that has been commonly used as a Western blotting and immunocytochemistry probe for TEM4 in mammalian cells. We find that this antibody, in addition to its intended target, cross-reacts with the Nuclear Mitotic Apparatus Protein 1 (NuMA) in Western blotting and immunoprecipitation, and detects NuMA preferentially in immunocytochemistry. This cross-reactivity, with an abundant chromatin- and mitotic spindle-associated factor, is likely to affect the interpretation of experiments that make use of this antibody probe, in particular by immunocytochemistry and immunoprecipitation.


Assuntos
Anticorpos , Proteínas de Ciclo Celular , Fatores de Troca de Nucleotídeo Guanina Rho , Fuso Acromático , Actinas/metabolismo , Animais , Anticorpos/imunologia , Proteínas de Ciclo Celular/imunologia , Mamíferos , Microtúbulos/metabolismo , Mitose , Fatores de Troca de Nucleotídeo Guanina Rho/imunologia , Fuso Acromático/metabolismo
7.
Int J Mol Sci ; 23(10)2022 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-35628256

RESUMO

The complete regression of clear cell renal cell carcinoma (ccRCC) obtained pre-clinically with anti-carbonic anhydrase IX (CAIX) G36 chimeric antigen receptor (CAR) T cells in doses equivalent to ≅108 CAR T cells/kg renewed the potential of this target to treat ccRCC and other tumors in hypoxia. The immune checkpoint blockade (ICB) brought durable clinical responses in advanced ccRCC and other tumors. Here, we tested CD8α/4-1BB compared to CD28-based anti-CAIX CAR peripheral blood mononuclear cells (PBMCs) releasing anti-programmed cell death ligand-1 (PD-L1) IgG4 for human ccRCC treatment in vitro and in an orthotopic NSG mice model in vivo. Using a ≅107 CAR PBMCs cells/kg dose, anti-CAIX CD28 CAR T cells releasing anti-PD-L1 IgG highly decrease both tumor volume and weight in vivo, avoiding the occurrence of metastasis. This antitumoral superiority of CD28-based CAR PBMCs cells compared to 4-1BB occurred under ICB via PD-L1. Furthermore, the T cell exhaustion status in peripheral CD4 T cells, additionally to CD8, was critical for CAR T cells efficiency. The lack of hepatotoxicity and nephrotoxicity upon the administration of a 107 CAR PMBCs cells/kg dose is the basis for carrying out clinical trials using anti-CAIX CD28 CAR PBMCs cells releasing anti-PD-L1 antibodies or anti-CAIX 4-1BB CAR T cells, offering exciting new prospects for the treatment of refractory ccRCC and hypoxic tumors.


Assuntos
Antígeno B7-H1 , Anidrase Carbônica IX , Carcinoma de Células Renais , Neoplasias Renais , Receptores de Antígenos Quiméricos , Animais , Anticorpos/imunologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Antígenos CD28 , Anidrase Carbônica IX/imunologia , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/terapia , Inibidores de Checkpoint Imunológico , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Leucócitos Mononucleares/patologia , Camundongos , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia
8.
J Clin Invest ; 132(12)2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35511419

RESUMO

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder whose most debilitating pathology is progressive and cumulative heterotopic ossification (HO) of skeletal muscles, ligaments, tendons, and fascia. FOP is caused by mutations in the type I BMP receptor gene ACVR1, which enable ACVR1 to utilize its natural antagonist, activin A, as an agonistic ligand. The physiological relevance of this property is underscored by the fact that HO in FOP is exquisitely dependent on activation of FOP-mutant ACVR1 by activin A, an effect countered by inhibition of anti-activin A via monoclonal antibody treatment. Hence, we surmised that anti-ACVR1 antibodies that block activation of ACVR1 by ligands should also inhibit HO in FOP and provide an additional therapeutic option for this condition. Therefore, we generated anti-ACVR1 monoclonal antibodies that block ACVR1's activation by its ligands. Surprisingly, in vivo, these anti-ACVR1 antibodies stimulated HO and activated signaling of FOP-mutant ACVR1. This property was restricted to FOP-mutant ACVR1 and resulted from anti-ACVR1 antibody-mediated dimerization of ACVR1. Conversely, wild-type ACVR1 was inhibited by anti-ACVR1 antibodies. These results uncover an additional property of FOP-mutant ACVR1 and indicate that anti-ACVR1 antibodies should not be considered as therapeutics for FOP.


Assuntos
Miosite Ossificante , Ossificação Heterotópica , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/farmacologia , Anticorpos/imunologia , Humanos , Ligantes , Mutação , Miosite Ossificante/genética , Ossificação Heterotópica/genética , Ossificação Heterotópica/patologia , Transdução de Sinais/genética
9.
Mol Neurobiol ; 59(7): 3980-3995, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35460053

RESUMO

Spreading of alpha-synuclein (αSyn) may play an important role in Parkinson's disease and related synucleinopathies. Passive immunization with anti-αSyn antibodies is a promising method to slow down the spreading process and thereby the progression of synucleinopathies. Currently, it remains elusive which specific characteristics are essential to render therapeutic antibodies efficacious. Here, we established a neuronal co-culture model, in which αSyn species are being released from αSyn-overexpressing cells and induce toxicity in a priori healthy GFP-expressing cells. In this model, we investigated the protective efficacy of three anti-αSyn antibodies. Only two of these antibodies, one C-terminal and one N-terminal, protected from αSyn-induced toxicity by inhibiting the uptake of spreading-competent αSyn from the cell culture medium. Neither the binding epitope nor the affinity of the antibodies towards recombinant αSyn could explain differences in biological efficacy. However, both protective antibodies formed more stable antibody-αSyn complexes than the non-protective antibody. These findings indicate that the stability of antibody-αSyn complexes may be more important to confer protection than the binding epitope or affinity to recombinant αSyn.


Assuntos
Anticorpos , Doença de Parkinson , Sinucleinopatias , alfa-Sinucleína , Anticorpos/imunologia , Anticorpos/farmacologia , Epitopos/imunologia , Humanos , Neurônios , Doença de Parkinson/imunologia , Doença de Parkinson/terapia , Sinucleinopatias/imunologia , Sinucleinopatias/terapia , alfa-Sinucleína/imunologia
10.
Front Immunol ; 13: 833715, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35242137

RESUMO

2020 will be marked in history for the dreadful implications of the COVID-19 pandemic that shook the world globally. The pandemic has reshaped the normality of life and affected mankind in the aspects of mental and physical health, financial, economy, growth, and development. The focus shift to COVID-19 has indirectly impacted an existing air-borne disease, Tuberculosis. In addition to the decrease in TB diagnosis, the emergence of the TB/COVID-19 syndemic and its serious implications (possible reactivation of latent TB post-COVID-19, aggravation of an existing active TB condition, or escalation of the severity of a COVID-19 during TB-COVID-19 coinfection), serve as primary reasons to equally prioritize TB. On a different note, the valuable lessons learnt for the COVID-19 pandemic provide useful knowledge for enhancing TB diagnostics and therapeutics. In this review, the crucial need to focus on TB amid the COVID-19 pandemic has been discussed. Besides, a general comparison between COVID-19 and TB in the aspects of pathogenesis, diagnostics, symptoms, and treatment options with importance given to antibody therapy were presented. Lastly, the lessons learnt from the COVID-19 pandemic and how it is applicable to enhance the antibody-based immunotherapy for TB have been presented.


Assuntos
Anticorpos/uso terapêutico , COVID-19/epidemiologia , COVID-19/terapia , Coinfecção/terapia , Tuberculose/epidemiologia , Tuberculose/terapia , Anticorpos/imunologia , COVID-19/diagnóstico , COVID-19/imunologia , Coinfecção/diagnóstico , Coinfecção/epidemiologia , Coinfecção/imunologia , Humanos , Imunoterapia , Mycobacterium tuberculosis , Receptores de Antígenos de Linfócitos T/imunologia , SARS-CoV-2/imunologia , Tuberculose/diagnóstico , Tuberculose/imunologia
11.
Proc Natl Acad Sci U S A ; 119(10): e2117034119, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35235454

RESUMO

Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease of the central nervous system (CNS) with a high socioeconomic relevance. The pathophysiology of MS, which is both complex and incompletely understood, is believed to be influenced by various environmental determinants, including diet. Since the 1990s, a correlation between the consumption of bovine milk products and MS prevalence has been debated. Here, we show that C57BL/6 mice immunized with bovine casein developed severe spinal cord pathology, in particular, demyelination, which was associated with the deposition of immunoglobulin G. Furthermore, we observed binding of serum from casein-immunized mice to mouse oligodendrocytes in CNS tissue sections and in culture where casein-specific antibodies induced complement-dependent pathology. We subsequently identified myelin-associated glycoprotein (MAG) as a cross-reactive antigenic target. The results obtained from the mouse model were complemented by clinical data showing that serum samples from patients with MS contained significantly higher B cell and antibody reactivity to bovine casein than those from patients with other neurologic diseases. This reactivity correlated with the B cell response to a mixture of CNS antigens and could again be attributed to MAG reactivity. While we acknowledge disease heterogeneity among individuals with MS, we believe that consumption of cow's milk in a subset of patients with MS who have experienced a previous loss of tolerance to bovine casein may aggravate the disease. Our data suggest that patients with antibodies to bovine casein might benefit from restricting dairy products from their diet.


Assuntos
Anticorpos/imunologia , Caseínas/imunologia , Reações Cruzadas , Doenças Desmielinizantes/imunologia , Esclerose Múltipla/imunologia , Glicoproteína Associada a Mielina/imunologia , Animais , Especificidade de Anticorpos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Leite/imunologia
13.
BMC Nephrol ; 23(1): 91, 2022 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-35247969

RESUMO

BACKGROUND: Antibody-mediated rejection (AMR) was described in kidney transplant patients after viral infections, such as the cytomegalovirus. Very few cases were recently reported after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, probably in the context of lowering of immunosuppressive therapy. To date, no direct immunological link was proved to explain a connection between the coronavirus disease 19 (COVID-19) infection and antibody-mediated rejection (AMR) if it exists. CASE PRESENTATION: Here we try to find this association by presenting the case of a low immunological risk patient who presented, six years post-transplant, with c4d negative antibody mediated rejection due to an anti-HLA-C17 de novo donor specific antibody (DSA) after contracting the coronavirus disease 19. The HLA-Cw17 activated the antibody-dependent cell-mediated cytotoxicity via the KIR2DS1 positive NK cells. DISCUSSION AND CONCLUSIONS: This case report may prove a direct role for COVID-19 infection in AMRs in the kidney transplant recipients, leading us to closely monitor kidney transplant recipients, especially if they have "at-risk" donor antigens.


Assuntos
Anticorpos/imunologia , COVID-19/complicações , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/imunologia , Transplante de Rim , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo
14.
Front Immunol ; 13: 823204, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35140723

RESUMO

The therapeutic efficacy of B-cell depletion by anti-CD20 treatment in pediatric and, more recently, in adult idiopathic nephrotic syndrome patients suggests a key role of B cells in the pathogenesis of the disease. However, their exact role is still unclear. B cells are able to secrete a large variety of antibodies that can protect against infections. However, B-cell dysregulation is well-established in a variety of autoimmune diseases. In parallel with their ability to produce antibodies, pathogenic B cells display altered effector functions by expressing activating surface molecules, which can strongly modify the immune homeostasis, or by producing specific cytokines, which can directly affect either podocyte structure and functions or modulate T-cell homeostasis. Herein, we report the most relevant clinical and experimental evidences of a pathogenic role of B cells in idiopathic nephrotic syndrome. We further highlight similarities and differences between children and adults affected by non-genetic forms of the disease and discuss what needs to be investigated in order to define the exact mechanisms underlying the pathogenic role of B cells and to identify more tailored therapeutic approaches.


Assuntos
Linfócitos B/patologia , Glomerulosclerose Segmentar e Focal/patologia , Nefrose Lipoide/patologia , Síndrome Nefrótica/patologia , Adulto , Anticorpos/imunologia , Linfócitos B/classificação , Linfócitos B/imunologia , Criança , Citocinas/imunologia , Glomerulosclerose Segmentar e Focal/imunologia , Humanos , Nefrose Lipoide/imunologia , Síndrome Nefrótica/imunologia , Fenótipo , Podócitos/patologia
15.
Int J Mol Sci ; 23(3)2022 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-35163258

RESUMO

Antibody-enzyme complexes (AECs) are ideal molecular recognition elements for immunosensing applications. One molecule possesses both a binding ability to specific targets and catalytic activity to gain signals, particularly oxidoreductases, which can be integrated into rapid and sensitive electrochemical measurements. The development of AECs using fragment antibodies rather than intact antibodies, such as immunoglobulin G (IgG), has attracted attention for overcoming the ethical and cost issues associated with the production of intact antibodies. Conventionally, chemical conjugation has been used to fabricate AECs; however, controlling stoichiometric conjugation using this method is difficult. To prepare homogeneous AECs, methods based on direct fusion and enzymatic conjugation have been developed, and more convenient methods using Catcher/Tag systems as coupling modules have been reported. In this review, we summarize the methods for fabricating AECs using fragment antibodies developed for sensing applications and discuss the advantages and disadvantages of each method.


Assuntos
Anticorpos/imunologia , Imunoensaio/métodos , Complexos Multienzimáticos/imunologia , Animais , Humanos , Imunoglobulina G/imunologia
16.
Cell Rep Med ; 3(1): 100499, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-35106511

RESUMO

Borna disease virus 1 (BoDV-1) causes rare but often fatal encephalitis in humans. Late diagnosis prohibits an experimental therapeutic approach. Here, we report a recent case of fatal BoDV-1 infection diagnosed on day 12 after hospitalization by detection of BoDV-1 RNA in the cerebrospinal fluid. In a retrospective analysis, we detect BoDV-1 RNA 1 day after hospital admission when the cell count in the cerebrospinal fluid is still normal. We develop a new ELISA using recombinant BoDV-1 nucleoprotein, phosphoprotein, and accessory protein X to detect seroconversion on day 12. Antibody responses are also shown in seven previously confirmed cases. The individual BoDV-1 antibody profiles show variability, but the usage of three different BoDV-1 antigens results in a more sensitive diagnostic tool. Our findings demonstrate that early detection of BoDV-1 RNA in cerebrospinal fluid and the presence of antibodies against at least two different viral antigens contribute to BoDV-1 diagnosis. Physicians in endemic regions should consider BoDV-1 infection in cases of unclear encephalopathy and initiate appropriate diagnostics at an early stage.


Assuntos
Anticorpos/imunologia , Doença de Borna/diagnóstico , Doença de Borna/imunologia , Vírus da Doença de Borna/fisiologia , Nucleoproteínas/imunologia , Fosfoproteínas/imunologia , Proteínas Virais/imunologia , Idoso , Animais , Chlorocebus aethiops , Humanos , Proteínas Recombinantes/imunologia , Células Vero
17.
Int J Mol Sci ; 23(3)2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35163118

RESUMO

G protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This results in the recruitment of arrestins, leading to desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffolding partners. GRKs' versatility is also reflected by their diverse roles in pathological conditions such as cancer, malaria, Parkinson's-, cardiovascular-, and metabolic disease. Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRKs (GRK2, GRK3, GRK5, and GRK6) in Western blot analysis. We identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross-reactivity. Hence, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained Western blot results. Utilizing the most-suitable antibodies, we established the Western blot-based, cost-effective simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in nine commonly used cell lines, revealing differential isoform expression.


Assuntos
Anticorpos/imunologia , Western Blotting/métodos , Quinases de Receptores Acoplados a Proteína G/análise , Quinases de Receptores Acoplados a Proteína G/metabolismo , Animais , Células CHO , Cricetulus , Quinases de Receptores Acoplados a Proteína G/imunologia , Células HEK293 , Humanos , Isoenzimas , Camundongos , Células NIH 3T3 , Fosforilação , Ratos , Transdução de Sinais
18.
Georgian Med News ; (322): 115-121, 2022 Jan.
Artigo em Russo | MEDLINE | ID: mdl-35134772

RESUMO

Aim - to identify deviations in immune parameters in patients with reactive chlamydial spondyloarthritis, allowing more targeted correction of immune status and improve the quality of treatment of these patients. A comparative immunological examination of 14 patients with reactive spondyloarthritis of chlamydial etiology before and after specific treatment and practically healthy people was carried out. CIC, lymphocytotoxic and granulocytotoxic antibodies, including autoimmune, LIF including autoantigens (cartilage, bone, sinewy), neutrophilic leukocytes, lymphocytes, IL-1, IL-4, IL-6, TNF-α, CD-3, CD-4, CD-8, CD-25, Ig A, G, M. The immune status of patients before treatment was characterized by a perverse immune response, which was characterized by hyperactivation of the T-lymphocytic link of immunity, impaired suppressive link of the immune system, a tendency to autoimmune aggression, incomplete anti-infectious immunity, and chronic inflammatory process. Antichlamydial treatment with the eradication of the pathogen within 1 month led to a partial normalization of the immune response, normalization of the neutrophilic / lymphocytic ratio and the amount of granulocytotoxic and lymphocytotoxic antibodies. Desensitization of the organism to autoantigens (cartilage and synovial tissue) was observed. Comprehensive analysis of immunological parameters before and after treatment in patients with reactive spondyloarthritis of chlamydial etiology allows monitoring the effectiveness of the treatment and increases its effectiveness.


Assuntos
Infecções por Chlamydia/imunologia , Espondilartrite , Anticorpos/imunologia , Autoantígenos , Estudos de Casos e Controles , Infecções por Chlamydia/complicações , Citocinas/imunologia , Humanos , Espondilartrite/imunologia , Espondilartrite/microbiologia , Linfócitos T/citologia
19.
J Biol Chem ; 298(3): 101715, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35151691

RESUMO

Infection by the bacterium Bordetella pertussis continues to cause considerable morbidity and mortality worldwide. Many current acellular pertussis vaccines include the antigen pertactin, which has presumptive adhesive and immunomodulatory activities, but is rapidly lost from clinical isolates after the introduction of these vaccines. To better understand the contributions of pertactin antibodies to protection and pertactin's role in pathogenesis, we isolated and characterized recombinant antibodies binding four distinct epitopes on pertactin. We demonstrate that four of these antibodies bind epitopes that are conserved across all three classical Bordetella strains, and competition assays further showed that antibodies binding these epitopes are also elicited by B. pertussis infection of baboons. Surprisingly, we found that representative antibodies binding each epitope protected mice against experimental B. pertussis infection. A cocktail of antibodies from each epitope group protected mice against a subsequent lethal dose of B. pertussis and greatly reduced lung colonization levels after sublethal challenge. Each antibody reduced B. pertussis lung colonization levels up to 100-fold when administered individually, which was significantly reduced when antibody effector functions were impaired, with no antibody mediating antibody-dependent complement-induced lysis. These data suggest that antibodies binding multiple pertactin epitopes protect primarily by the same bactericidal mechanism, which overshadows contributions from blockade of other pertactin functions. These antibodies expand the available tools to further dissect pertactin's role in infection and understand the impact of antipertactin antibodies on bacterial fitness.


Assuntos
Anticorpos , Proteínas da Membrana Bacteriana Externa , Bordetella pertussis , Fatores de Virulência de Bordetella , Coqueluche , Animais , Anticorpos/imunologia , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Epitopos , Camundongos , Vacina contra Coqueluche/imunologia , Fatores de Virulência de Bordetella/química , Fatores de Virulência de Bordetella/imunologia , Fatores de Virulência de Bordetella/metabolismo , Coqueluche/prevenção & controle
20.
EMBO J ; 41(4): e109108, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35019161

RESUMO

Haploinsufficiency of the progranulin (PGRN)-encoding gene (GRN) causes frontotemporal lobar degeneration (GRN-FTLD) and results in microglial hyperactivation, TREM2 activation, lysosomal dysfunction, and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology, we used genetic and pharmacological approaches to suppress TREM2-dependent transition of microglia from a homeostatic to a disease-associated state. Trem2 deficiency in Grn KO mice reduced microglia hyperactivation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies led to reduced TREM2 signaling due to its enhanced shedding. Furthermore, TREM2 antibody-treated PGRN-deficient microglia derived from human-induced pluripotent stem cells showed reduced microglial hyperactivation, TREM2 signaling, and phagocytic activity, but lysosomal dysfunction was not rescued. Similarly, lysosomal dysfunction, lipid dysregulation, and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Synaptic loss and neurofilament light-chain (NfL) levels, a biomarker for neurodegeneration, were further elevated in the Grn/Trem2 KO cerebrospinal fluid (CSF). These findings suggest that TREM2-dependent microglia hyperactivation in models of GRN deficiency does not promote neurotoxicity, but rather neuroprotection.


Assuntos
Degeneração Lobar Frontotemporal/patologia , Glicoproteínas de Membrana/metabolismo , Microglia/fisiologia , Monócitos/metabolismo , Progranulinas/deficiência , Receptores Imunológicos/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Encéfalo/diagnóstico por imagem , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Feminino , Degeneração Lobar Frontotemporal/metabolismo , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Quinase Syk/metabolismo
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