RESUMO
OBJECTIVE: Glycolytic inhibition via 2-deoxy-D-glucose (2DG) has potential therapeutic benefits for a range of diseases, including cancer, epilepsy, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA), and COVID-19, but the systemic effects of 2DG on gene function across different tissues are unclear. METHODS: This study analyzed the transcriptional profiles of nine tissues from C57BL/6J mice treated with 2DG to understand how it modulates pathways systemically. Principal component analysis (PCA), weighted gene co-network analysis (WGCNA), analysis of variance, and pathway analysis were all performed to identify modules altered by 2DG treatment. RESULTS: PCA revealed that samples clustered predominantly by tissue, suggesting that 2DG affects each tissue uniquely. Unsupervised clustering and WGCNA revealed six distinct tissue-specific modules significantly affected by 2DG, each with unique key pathways and genes. 2DG predominantly affected mitochondrial metabolism in the heart, while in the small intestine, it affected immunological pathways. CONCLUSIONS: These findings suggest that 2DG has a systemic impact that varies across organs, potentially affecting multiple pathways and functions. The study provides insights into the potential therapeutic benefits of 2DG across different diseases and highlights the importance of understanding its systemic effects for future research and clinical applications.
Assuntos
Desoxiglucose , Epilepsia , Camundongos , Animais , Desoxiglucose/farmacologia , Desoxiglucose/metabolismo , Camundongos Endogâmicos C57BL , Glucose/metabolismo , Perfilação da Expressão GênicaRESUMO
The Lamc2jeb junctional epidermolysis bullosa (EB) mouse model has been used to demonstrate that significant genetic modification of EB symptoms is possible, identifying as modifiers Col17a1 and six other quantitative trait loci, several with strong candidate genes including dystonin (Dst/Bpag1). Here, CRISPR/Cas9 was used to alter exon 23 in mouse skin specific isoform Dst-e (Ensembl GRCm38 transcript name Dst-213, transcript ID ENSMUST00000183302.5, protein size 2639AA) and validate a proposed arginine/glutamine difference at amino acid p1226 in B6 versus 129 mice as a modifier of EB. Frame shift deletions (FSD) in mouse Dst-e exon 23 (Dst-eFSD/FSD) were also identified that cause mice carrying wild-type Lamc2 to develop a phenotype similar to human EB simplex without dystonia musculorum. When combined, Dst-eFSD/FSD modifies Lamc2jeb/jeb (FSD+jeb) induced disease in unexpected ways implicating an altered balance between DST-e (BPAG1e) and a rarely reported rodless DST-eS (BPAG1eS) in epithelium as a possible mechanism. Further, FSD+jeb mice with pinnae removed are found to provide a test bed for studying internal epithelium EB disease and treatment without severe skin disease as a limiting factor while also revealing and accelerating significant nasopharynx symptoms present but not previously noted in Lamc2jeb/jeb mice.
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Distonia , Distúrbios Distônicos , Epidermólise Bolhosa Simples , Epidermólise Bolhosa Juncional , Epidermólise Bolhosa , Animais , Camundongos , Distonia/genética , Distonia/metabolismo , Distúrbios Distônicos/metabolismo , Distonina/metabolismo , Epidermólise Bolhosa/genética , Epidermólise Bolhosa Simples/diagnóstico , Epidermólise Bolhosa Simples/genética , Epidermólise Bolhosa Simples/metabolismo , Epidermólise Bolhosa Juncional/genética , Epidermólise Bolhosa Juncional/diagnóstico , Epidermólise Bolhosa Juncional/metabolismo , Pele/metabolismoRESUMO
Previous work strongly implicated Collagen 17a1 (Col17a1) as a potent genetic modifier of junctional epidermolysis bullosa (JEB) caused by a hypomorphic mutation (Lamc2jeb) in mice. The importance of the noncollagenous domain (NC4) of COLXVII was suggested by use of a congenic reduction approach that restricted the modifier effect to 2-3 neighboring amino acid changes in that domain. The current study utilizes TALEN and CRISPR/Cas9 induced amino acid replacements and in-frame indels nested to NC4 to further investigate the role of this and adjoining COLXVII domains both as modifiers and primary risk effectors. We confirm the importance of COLXVI AA 1275 S/G and 1277 N/S substitutions and utilize small nested indels to show that subtle changes in this microdomain attenuate JEB. We further show that large in-frame indels removing up to 1482 bp and 169 AA of NC6 through NC1 domains are surprisingly disease free on their own but can be very potent modifiers of Lamc2jeb/jeb JEB. Together these studies exploiting gene editing to functionally dissect the Col17a1 modifier demonstrate the importance of epistatic interactions between a primary disease-causing mutation in one gene and innocuous 'healthy' alleles in other genes.
Assuntos
Epidermólise Bolhosa Juncional , Animais , Camundongos , Epidermólise Bolhosa Juncional/genética , Colágenos não Fibrilares/genética , Colágenos não Fibrilares/metabolismo , Colágeno/genética , Mutação , Aminoácidos/genéticaRESUMO
Aberrant metabolic demand is observed in immune/inflammatory disorders, yet the role in pathogenesis remains unclear. Here, we discover that in lupus, activated B cells, including germinal center B (GCB) cells, have remarkably high glycolytic requirement for survival over T cell populations, as demonstrated by increased metabolic activity in lupus-activated B cells compared to immunization-induced cells. The augmented reliance on glucose oxidation makes GCB cells vulnerable to mitochondrial ROS-induced oxidative stress and apoptosis. Short-term glycolysis inhibition selectively reduces pathogenic activated B in lupus-prone mice, extending their lifespan, without affecting T follicular helper cells. Particularly, BCMA-expressing GCB cells rely heavily on glucose oxidation. Depleting BCMA-expressing activated B cells with APRIL-based CAR-T cells significantly prolongs the lifespan of mice with severe autoimmune disease. These results reveal that glycolysis-dependent activated B and GCB cells, especially those expressing BCMA, are potentially key lupus mediators, and could be targeted to improve disease outcomes.
RESUMO
Epidermolysis Bullosa (EB) is a group of rare genetic disorders that compromise the structural integrity of the skin such that blisters and subsequent erosions occur after minor trauma. While primary genetic risk of all subforms of EB adhere to Mendelian patterns of inheritance, their clinical presentations and severities can vary greatly, implying genetic modifiers. The Lamc2jeb mouse model of non-Herlitz junctional EB (JEB-nH) demonstrated that genetic modifiers can contribute substantially to the phenotypic variability of JEB and likely other forms of EB. The innocuous changes in an 'EB related gene', Col17a1, have shown it to be a dominant modifier of Lamc2jeb. This work identifies six additional Quantitative Trait Loci (QTL) that modify disease in Lamc2jeb/jeb mice. Three QTL include other known 'EB related genes', with the strongest modifier effect mapping to a region including the epidermal hemi-desmosomal structural gene dystonin (Dst-e/Bpag1-e). Three other QTL map to intervals devoid of known EB-associated genes. Of these, one contains the nuclear receptor coactivator Ppargc1a as its primary candidate and the others contain related genes Pparg and Igf1, suggesting modifier pathways. These results, demonstrating the potent disease modifying effects of normally innocuous genetic variants, greatly expand the landscape of genetic modifiers of EB and therapeutic approaches that may be applied.
Assuntos
Epidermólise Bolhosa Juncional , Animais , Camundongos , Pele , Vesícula , Epiderme , Locos de Características Quantitativas/genéticaRESUMO
OBJECTIVE: Glycolytic inhibition via 2-deoxy-D-glucose (2DG) has potential therapeutic benefits for a range of diseases, including cancer, epilepsy, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA), and COVID-19, but the systemic effects of 2DG on gene function across different tissues are unclear. METHODS: This study analyzed the transcriptional profiles of nine tissues from C57BL/6J mice treated with 2DG to understand how it modulates pathways systemically. Principal component analysis (PCA), weighted gene co-network analysis (WGCNA), analysis of variance, and pathway analysis were all performed to identify modules altered by 2DG treatment. RESULTS: PCA revealed that samples clustered predominantly by tissue, suggesting that 2DG affects each tissue uniquely. Unsupervised clustering and WGCNA revealed six distinct tissue-specific modules significantly affected by 2DG, each with unique key pathways and genes. 2DG predominantly affected mitochondrial metabolism in the heart, while in the small intestine, it affected immunological pathways. CONCLUSIONS: These findings suggest that 2DG has a systemic impact that varies across organs, potentially affecting multiple pathways and functions. The study provides insights into the potential therapeutic benefits of 2DG across different diseases and highlights the importance of understanding its systemic effects for future research and clinical applications.
RESUMO
BACKGROUND AND PURPOSE: Some epilepsy syndromes (sleep-related epilepsies, SREs) have a strong link with sleep. Comorbid sleep disorders are common in patients with SRE and can exert a negative impact on seizure control and quality of life. Our purpose was to define the standard procedures for the diagnostic pathway of patients with possible SRE (scenario 1) and the general management of patients with SRE and comorbidity with sleep disorders (scenario 2). METHODS: The project was conducted under the auspices of the European Academy of Neurology, the European Sleep Research Society and the International League Against Epilepsy Europe. The framework entailed the following phases: conception of the clinical scenarios; literature review; statements regarding the standard procedures. For the literature search a stepwise approach starting from systematic reviews to primary studies was applied. Published studies were identified from the National Library of Medicine's MEDLINE database and Cochrane Library. RESULTS: Scenario 1: Despite a low quality of evidence, recommendations on anamnestic evaluation and tools for capturing the event at home or in the laboratory are provided for specific SREs. Scenario 2: Early diagnosis and treatment of sleep disorders (especially respiratory disorders) in patients with SRE are likely to be beneficial for seizure control. CONCLUSIONS: Definitive procedures for evaluating patients with SRE are lacking. Advice is provided that could be of help for standardizing and improving the diagnostic approach of specific SREs. The importance of identifying and treating specific sleep disorders for the management and outcome of patients with SRE is underlined.
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Epilepsia Reflexa , Transtornos do Sono-Vigília , Consenso , Humanos , Qualidade de Vida , Sono , Transtornos do Sono-Vigília/diagnóstico , Transtornos do Sono-Vigília/epidemiologiaRESUMO
Needle-free uptake across mucosal barriers is a preferred route for delivery of biologics, but the efficiency of unassisted transmucosal transport is poor. To make administration and therapy efficient and convenient, strategies for the delivery of biologics must enhance both transcellular delivery and plasma half-life. We found that human albumin was transcytosed efficiently across polarized human epithelial cells by a mechanism that depends on the neonatal Fc receptor (FcRn). FcRn also transported immunoglobulin G, but twofold less than albumin. We therefore designed a human albumin variant, E505Q/T527M/K573P (QMP), with improved FcRn binding, resulting in enhanced transcellular transport upon intranasal delivery and extended plasma half-life of albumin in transgenic mice expressing human FcRn. When QMP was fused to recombinant activated coagulation factor VII, the half-life of the fusion molecule increased 3.6-fold compared with the wild-type human albumin fusion, without compromising the therapeutic properties of activated factor VII. Our findings highlight QMP as a suitable carrier of protein-based biologics that may enhance plasma half-life and delivery across mucosal barriers.
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Produtos Biológicos , Albumina Sérica Humana , Albuminas , Meia-Vida , Antígenos de Histocompatibilidade Classe I , Receptores Fc , Proteínas Recombinantes de FusãoRESUMO
IgG immune complexes (ICs) promote autoimmunity through binding fragment crystallizable (Fc) γ-receptors (FcγRs). Of these, the highly prevalent FcγRIIa (CD32a) histidine (H)-131 variant (CD32aH) is strongly linked to human autoimmune diseases through unclear mechanisms. We show that, relative to the CD32a arginine (R)-131 (CD32aR) variant, CD32aH more avidly bound human (h) IgG1 IC and formed a ternary complex with the neonatal Fc receptor (FcRn) under acidic conditions. In primary human and mouse cells, both CD32a variants required FcRn to induce innate and adaptive immune responses to hIgG1 ICs, which were augmented in the setting of CD32aH. Conversely, FcRn induced responses to IgG IC independently of classical FcγR, but optimal responses required FcRn and FcγR. Finally, FcRn blockade decreased inflammation in a rheumatoid arthritis model without reducing circulating autoantibody levels, providing support for FcRn's direct role in IgG IC-associated inflammation. Thus, CD32a and FcRn coregulate IgG IC-mediated immunity in a manner favoring the CD32aH variant, providing a novel mechanism for its disease association.
Assuntos
Autoimunidade/imunologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Imunoglobulina G/imunologia , Receptores Fc/fisiologia , Imunidade Adaptativa/imunologia , Animais , Artrite Reumatoide/imunologia , Suscetibilidade a Doenças , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunidade Inata/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Fc/imunologia , Receptores de IgG/imunologiaRESUMO
Albumin has an average plasma half-life of three weeks and is thus an attractive carrier to improve the pharmacokinetics of fused therapeutics. The half-life is regulated by FcRn, a cellular receptor that protects against intracellular degradation. To tailor-design the therapeutic use of albumin, it is crucial to understand how structural alterations in albumin affect FcRn binding and transport properties. In the blood, the last C-terminal residue (L585) of albumin may be enzymatically cleaved. Here we demonstrate that removal of the L585 residue causes structural stabilization in regions of the principal FcRn binding domain and reduces receptor binding. In line with this, a short half-life of only 3.5 days was measured for cleaved albumin lacking L585 in a patient with acute pancreatitis. Thus, we reveal the structural requirement of an intact C-terminal end of albumin for a long plasma half-life, which has implications for design of albumin-based therapeutics.
Assuntos
Albumina Sérica Humana/metabolismo , Amilases/sangue , Animais , Carboxipeptidases A/sangue , Meia-Vida , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Lipase/sangue , Masculino , Camundongos Transgênicos , Pâncreas/enzimologia , Pancreatite/sangue , Pancreatite/enzimologia , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Proteólise , Receptores Fc/genética , Receptores Fc/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Albumina Sérica Humana/química , Albumina Sérica Humana/genética , Relação Estrutura-AtividadeRESUMO
Almost a decade has passed since the approval of belimumab, an mAb directed against B lymphocyte stimulation and the first targeted therapy approved for systemic lupus erythematous (SLE) in over 50 y. Although well tolerated, the efficacy of belimumab remains limited and is not labeled for patients suffering from nephritis, the leading cause of patient mortality. We sought to explore alternative targets of autoreactive B lymphocytes through manipulation of affinity maturation. The BXSB/MpJ mouse, a well-established model of human SLE, develops elevated antinuclear Abs and immune complex-mediated nephritis along with other manifestations of SLE-like disease. To limit interfering with critical background genetics, we used CRISPR-Cas9 to disrupt activation-induced cytidine deaminase (AID; Aicda) directly in BXSB zygotes. Homozygous null mice demonstrated significantly prolonged survival compared with wild-type. Although mice continued to develop plasma cells, splenic follicular structure was restored, and renal pathology was reduced. Mice developed expanded germinal center B lymphocyte populations as in other models of AID deficiency as well as increased populations of CD73+ B lymphocytes. Treatment with the small molecule inhibitor of RAD51, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, resulted in minimal changes in disease markers in BXSB mice. The prolonged survival in AID-deficient BXSB mice appears attributed primarily to the reduced renal pathology, warranting further exploration, as current therapeutics targeting lupus nephritis are limited and, thus, in great demand.
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Subpopulações de Linfócitos B/imunologia , Citidina Desaminase/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Animais , Subpopulações de Linfócitos B/patologia , Sistemas CRISPR-Cas , Citidina Desaminase/genética , Modelos Animais de Doenças , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Camundongos KnockoutRESUMO
2-deoxy D-glucose (2DG) was tested for efficacy in treating alopecia areata using the C3H/HeJ skin graft model. 2DG has proven to be efficacious in treatment of various mouse models of autoimmunity with minimal serious side effects noted. This agent has been shown to normalize abnormally activated T-cell populations while also preventing cell surface expression of NKG2D; key factors defining alopecia areata disease progression. Daily oral ingestion of 2DG via drinking water to mice with patchy or diffuse alopecia areata for 16 weeks failed to prevent expansion of alopecia or cause regrowth of hair in treated mice. Histologically, there were no differences between treated and control groups. These results indicate that, while 2DG is effective for some autoimmune diseases, it was not efficacious for the cell-mediated autoimmune mouse disease, alopecia areata.
Assuntos
Alopecia em Áreas/tratamento farmacológico , Desoxiglucose/uso terapêutico , Animais , Desoxiglucose/administração & dosagem , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Folículo Piloso/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C3H , Transplante de Pele , Falha de TratamentoRESUMO
BACKGROUND AND PURPOSE: The aim of this study was to identify the prevalence of sleep disorders and measure the objective sleep quality in patients with seizure disorders. METHODS: Patients admitted for video electroencephalography monitoring were prospectively recruited and polysomnography was performed on the third night of monitoring. RESULTS: A total of 4/44 (9%) patients with epilepsy and 2/22 (9%) patients with dissociative seizures were found to have mild sleep-disordered breathing. Three (7%) patients with epilepsy were found to have mild or moderate obstructive sleep apnea-hypopnea syndrome (OSAHS) and three (14%) patients with dissociative seizures had mild or moderate OSAHS. Most patients with sleep-disordered breathing or OSAHS were overweight or obese. Time awake after sleep onset was high in both groups. There were no significant differences in sleep architecture between the groups except for a difference in average N3 sleep stage proportion. Periodic limb movements (PLMs) were common in both groups and 27% of patients with dissociative seizures had both high PLM rates and high arousal indices, suggesting a high prevalence of probable PLM disorder in that group (compared with 9% in the epilepsy group). CONCLUSIONS: Our findings contradict the commonly reported high comorbidity of OSAHS and epilepsy, and question its purported clinical relevance. High rates of PLMs were found in patients with dissociative seizures. In both patient groups, high awake after sleep onset times were indicative of sleep disruption, which can have an epileptogenic effect and is known to increase dissociative tendencies.
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Epilepsia/fisiopatologia , Convulsões/fisiopatologia , Transtornos do Sono-Vigília/fisiopatologia , Sono/fisiologia , Adulto , Epilepsia/complicações , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polissonografia , Convulsões/complicações , Transtornos do Sono-Vigília/complicações , VigíliaRESUMO
Targeting the early steps of the glycolysis pathway in cancers is a well-established therapeutic strategy; however, the doses required to elicit a therapeutic effect on the cancer can be toxic to the patient. Consequently, numerous preclinical and clinical studies have combined glycolytic blockade with other therapies. However, most of these other therapies do not specifically target cancer cells, and thus adversely affect normal tissue. Here we first show that a diverse number of cancer models - spontaneous, patient-derived xenografted tumor samples, and xenografted human cancer cells - can be efficiently targeted by 2-deoxy-D-Glucose (2DG), a well-known glycolytic inhibitor. Next, we tested the cancer-cell specificity of a therapeutic compound using the MEC1 cell line, a chronic lymphocytic leukemia (CLL) cell line that expresses activation induced cytidine deaminase (AID). We show that MEC1 cells, are susceptible to 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), a specific RAD51 inhibitor. We then combine 2DG and DIDS, each at a lower dose and demonstrate that this combination is more efficacious than fludarabine, the current standard- of- care treatment for CLL. This suggests that the therapeutic blockade of glycolysis together with the therapeutic inhibition of RAD51-dependent homologous recombination can be a potentially beneficial combination for targeting AID positive cancer cells with minimal adverse effects on normal tissue. Implications: Combination therapy targeting glycolysis and specific RAD51 function shows increased efficacy as compared to standard of care treatments in leukemias.
Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Desoxiglucose/farmacologia , Neoplasias/tratamento farmacológico , Rad51 Recombinase/antagonistas & inibidores , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/administração & dosagem , Animais , Linhagem Celular Tumoral , Desoxiglucose/administração & dosagem , Sinergismo Farmacológico , Feminino , Glicólise/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Neoplasias/metabolismo , Rad51 Recombinase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Follicular helper T (TFH) cells are expanded in systemic lupus erythematosus, where they are required to produce high affinity autoantibodies. Eliminating TFH cells would, however compromise the production of protective antibodies against viral and bacterial pathogens. Here we show that inhibiting glucose metabolism results in a drastic reduction of the frequency and number of TFH cells in lupus-prone mice. However, this inhibition has little effect on the production of T-cell-dependent antibodies following immunization with an exogenous antigen or on the frequency of virus-specific TFH cells induced by infection with influenza. In contrast, glutaminolysis inhibition reduces both immunization-induced and autoimmune TFH cells and humoral responses. Solute transporter gene signature suggests different glucose and amino acid fluxes between autoimmune TFH cells and exogenous antigen-specific TFH cells. Thus, blocking glucose metabolism may provide an effective therapeutic approach to treat systemic autoimmunity by eliminating autoreactive TFH cells while preserving protective immunity against pathogens.
Assuntos
Glucose/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Aminoácidos/metabolismo , Animais , Autoanticorpos/metabolismo , Autoimunidade/fisiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , CamundongosRESUMO
The K/BxN mouse is a spontaneous model of arthritis driven by T cell receptor transgenic CD4+ T cells from the KRN strain that are activated by glucose-6-phosphate isomerase (GPI) peptides presented by the H-2g7 allele from the NOD strain. It is a model of autoimmune seropositive arthritis because the production of anti-GPI IgG is necessary and sufficient for joint pathology. The production of high levels of anti-GPI IgG requires on the expansion of CD4+ follicular helper T (Tfh) cells. The metabolic requirements of this expansion have never been characterized. Based on the therapeutic effects of the combination of metformin and 2-deoxyglucose (2DG) in lupus models that normalized the expansion of effector CD4+ T cells. We showed that the CD4+ T cells and to a lesser extent, the B cells from K/BxN mice are more metabolically active than the KRN controls. Accordingly, preventive inhibition of glycolysis with 2DG significantly reduced joint inflammation and the activation of both adaptive and innate immune cells, as well as the production of pathogenic autoantibodies. However, contrary to the lupus-prone mice, the addition of metformin had little beneficial effect, suggesting that glycolysis is the major driver of immune activation in this model. We propose that K/BxN mice are another model in which autoreactive Tfh cells are highly glycolytic and that their function can be limited by inhibiting glucose metabolism.
Assuntos
Artrite Reumatoide/metabolismo , Desoxiglucose/administração & dosagem , Glicólise/fisiologia , Articulações/imunologia , Animais , Artrite Reumatoide/imunologia , Autoanticorpos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Progressão da Doença , Centro Germinativo/imunologia , Glucose-6-Fosfato Isomerase/imunologia , Humanos , Metformina/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
The neonatal Fc receptor (FcRn) has been demonstrated to contribute to a high bioavailability of monoclonal antibodies (mAbs). In this study, we explored the cellular sites of FcRn-mediated protection after subcutaneous (SC) and intravenous (IV) administration. SC absorption and IV disposition kinetics of a mAb were studied in hFcRn transgenic (Tg) bone marrow chimeric mice in which hFcRn was restricted to radioresistant cells or hematopoietic cells. SC bioavailabilities close to 90% were observed in hFcRn Tg mice and chimeric mice with hFcRn expression in hematopoietic cells, whereas SC bioavailabilities were markedly lower when FcRn was missing in hematopoietic cells. Our study demonstrates: 1) FcRn in radiosensitive hematopoietic cells is required for high SC bioavailability, indicating first-pass catabolism after SC administration by hematopoietic cells; 2) FcRn-mediated transcytosis or recycling by radioresistent cells is not required for high SC bioavailability; and 3) after IV administration hematopoietic and radioresistent cells contribute about equally to clearance of the mAb. A pharmacokinetic model was devised to describe a mixed elimination via radioresistent and hematopoietic cells from vascular and extravascular compartments, respectively. Overall, the study indicates a relevant role of hematopoietic cells for first-pass clearance of mAbs after SC administration and confirms their role in the overall clearance of mAbs.
Assuntos
Anticorpos Monoclonais/farmacocinética , Células-Tronco Hematopoéticas/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores Fc/metabolismo , Administração Intravenosa , Animais , Anticorpos Monoclonais/administração & dosagem , Disponibilidade Biológica , Transplante de Células-Tronco Hematopoéticas , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Injeções Subcutâneas , Taxa de Depuração Metabólica , Camundongos , Camundongos Transgênicos , Receptores Fc/genéticaRESUMO
Mouse models of lupus have shown that multiple immune cell types contribute to autoimmune disease. This study sought to investigate the involvement of B cells and dendritic cells in supporting the expansion of inflammatory and regulatory CD4+ T cells that are critical for lupus pathogenesis. We used lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) and congenic C57BL/6J (B6) control mice to investigate how the genetic predisposition of these two cell types controls the activity of normal B6 T cells. Using an allogeneic in vitro assay, we showed that TC B1-a and conventional B cells expanded Th17 cells significantly more than their B6 counterparts. This expansion was dependent on CD86 and IL-6 expression and mapped to the Sle1 lupus-susceptibility locus. In vivo, TC B cells promoted greater differentiation of CD4+ T cells into Th1 and follicular helper T cells than did B6 B cells, but they limited the expansion of Foxp3 regulatory CD4+ T cells to a greater extent than did B6 B cells. Finally, when normal B6 CD4+ T cells were introduced into Rag1-/- mice, TC myeloid/stromal cells caused their heightened activation, decreased Foxp3 regulatory CD4+ T cell differentiation, and increased renal infiltration of Th1 and Th17 cells in comparison with B6 myeloid/stromal cells. The results show that B cells from lupus mice amplify inflammatory CD4+ T cells in a nonredundant manner with myeloid/stromal cells.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária/imunologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Albumin and IgG have remarkably long serum half-lives due to pH-dependent FcRn-mediated cellular recycling that rescues both ligands from intracellular degradation. Furthermore, increase in half-lives of IgG and albumin-based therapeutics has the potential to improve their efficacies, but there is a great need for robust methods for screening of relative FcRn-dependent recycling ability. Here, we report on a novel human endothelial cell-based recycling assay (HERA) that can be used for such pre-clinical screening. In HERA, rescue from degradation depends on FcRn, and engineered ligands are recycled in a manner that correlates with their half-lives in human FcRn transgenic mice. Thus, HERA is a novel cellular assay that can be used to predict how FcRn-binding proteins are rescued from intracellular degradation.