RESUMO
The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR-targeting immunotoxin consisting of a clinically proven anti-EGFR IgG (cetuximab; CTX) and a toxin fragment (LR-LO10) derived from Pseudomonas exotoxin A (PE) using a novel site-specific conjugation technology (peptide-directed photo-crosslinking reaction), as an alternative option. The immunotoxin (CTX-LR-LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor-2. Treatment of EGFR-positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX-LR-LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR-LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX-LR-LO10 may serve as a new therapeutic agent targeting EGFR-positive cancers.
Assuntos
ADP Ribose Transferases , Receptores ErbB , Exotoxinas , Neoplasias de Cabeça e Pescoço , Imunoglobulina G , Imunotoxinas , Exotoxina A de Pseudomonas aeruginosa , Fatores de Virulência , Humanos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Receptores ErbB/imunologia , Animais , Imunotoxinas/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Camundongos , Imunoglobulina G/farmacologia , Linhagem Celular Tumoral , Exotoxinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Cetuximab/farmacologia , Camundongos Nus , Toxinas Bacterianas , Apoptose/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Feminino , Movimento Celular/efeitos dos fármacos , Antineoplásicos/farmacologiaRESUMO
The relevance of aberrant serum IgG N-glycosylation in liver fibrosis has been identified; however, its causal effect remains unclear. Because hepatic stellate cells (HSCs) contribute substantially to liver fibrosis, we investigated whether and through which mechanisms IgG N-glycosylation affects the fibrogenic properties of HSCs. Analysis of serum IgG1 N-glycome from 151 patients with chronic hepatitis B or liver cirrhosis revealed a positive correlation between Ishak fibrosis grading and IgG1 with agalactosyl N-glycoforms on the crystallizable fragment (Fc). Fc gamma receptor (FcγR) IIIa was observed in cultured human HSCs and HSCs in human liver tissues, and levels of FcγRIIIa in HSCs correlated with the severity of liver fibrosis. Additionally, agalactosyl IgG treatment caused HSCs to have a fibroblast-like morphology, enhanced migration and invasion capabilities, and enhanced expression of the FcγRIIIa downstream tyrosine-protein kinase SYK. Furthermore, agalactosyl IgG treatment increased fibrogenic factors in HSCs, including transforming growth factor (TGF)-ß1, total collagen, platelet-derived growth factor subunit B and its receptors, pro-collagen I-α1, α-smooth muscle actin, and matrix metalloproteinase 9. These effects were more pronounced in HSCs that stably expressed FCGR3A and were reduced in FCGR3A knockout cells. Agalactosyl IgG and TGF-ß1 each increased FCGR3A in HSCs. Furthermore, serum TGF-ß1 concentrations in patients were positively correlated with agalactosyl IgG1 levels and liver fibrosis severity, indicating a positive feedback loop involving agalactosyl IgG, HSC-FcγRIIIa, and TGF-ß1. In conclusion, agalactosyl IgG promotes fibrogenic characteristics in HSCs through FcγRIIIa. © 2024 The Pathological Society of Great Britain and Ireland.
Assuntos
Células Estreladas do Fígado , Imunoglobulina G , Cirrose Hepática , Receptores de IgG , Humanos , Receptores de IgG/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Glicosilação , Masculino , Pessoa de Meia-Idade , Feminino , Movimento Celular , Hepatite B Crônica/patologia , Hepatite B Crônica/metabolismo , Transdução de Sinais , Quinase Syk/metabolismo , Adulto , Idoso , Células CultivadasRESUMO
Anti-ganglioside antibodies (anti-Gg Abs) have been linked to delayed/poor clinical recovery in both axonal and demyelinating forms of Guillain-Barrè Syndrome (GBS). In many instances, the incomplete recovery is attributed to the peripheral nervous system's failure to regenerate. The cross-linking of cell surface gangliosides by anti-Gg Abs triggers inhibition of nerve repair in both in vitro and in vivo axon regeneration paradigms. This mechanism involves the activation of the small GTPase RhoA, which negatively modulates the growth cone cytoskeleton. At present, the identity/es of the receptor/s responsible for transducing the signal that ultimately leads to RhoA activation remains poorly understood. The aim of this work was to identify the transducer molecule responsible for the inhibitory effect of anti-Gg Abs on nerve repair. Putative candidate molecules were identified through proteomic mass spectrometry of ganglioside affinity-captured proteins from rat cerebellar granule neurons (Prendergast et al., 2014). These candidates were evaluated using an in vitro model of neurite outgrowth with primary cultured dorsal root ganglion neurons (DRGn) and an in vivo model of axon regeneration. Using an shRNA-strategy to silence putative candidates on DRGn, we identified tumor necrosis factor receptor 1A protein (TNFR1A) as a transducer molecule for the inhibitory effect on neurite outgrowth from rat/mouse DRGn cultures of a well characterized mAb targeting the related gangliosides GD1a and GT1b. Interestingly, lack of TNFr1A expression on DRGn abolished the inhibitory effect on neurite outgrowth caused by anti-GD1a but not anti-GT1b specific mAbs, suggesting specificity of GD1a/transducer signaling. Similar results were obtained using primary DRGn cultures from TNFR1a-null mice, which did not activate RhoA after exposure to anti-GD1a mAbs. Generation of single point mutants at the stalk region of TNFR1A identified a critical amino acid for transducing GD1a signaling, suggesting a direct interaction. Finally, passive immunization with an anti-GD1a/GT1b mAb in an in vivo model of axon regeneration exhibited reduced inhibitory activity in TNFR1a-null mice compared to wild type mice. In conclusion, these findings identify TNFR1A as a novel transducer receptor for the inhibitory effect exerted by anti-GD1a Abs on nerve repair, representing a significant step forward toward understanding the factors contributing to poor clinical recovery in GBS associated with anti-Gg Abs.
Assuntos
Axônios , Gangliosídeos , Imunoglobulina G , Regeneração Nervosa , Receptores Tipo I de Fatores de Necrose Tumoral , Proteína rhoA de Ligação ao GTP , Animais , Gangliosídeos/metabolismo , Gangliosídeos/imunologia , Axônios/metabolismo , Axônios/imunologia , Ratos , Camundongos , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Síndrome de Guillain-Barré/imunologia , Síndrome de Guillain-Barré/metabolismo , Síndrome de Guillain-Barré/patologia , Camundongos Knockout , Transdução de Sinais , Células CultivadasRESUMO
OBJECTIVES: A positive allosteric modulator of the NMDAR, SGE-301, has been shown to reverse the alterations caused by the antibodies of patients with anti-NMDAR encephalitis (NMDARe). However, the mechanisms involved beyond receptor modulation are unclear. In this study, we aimed to investigate how this modulator affects NMDAR membrane dynamics. METHODS: Cultured hippocampal neurons were treated with SGE-301 or vehicle, alongside with immunoglobulins G (IgG) from patients with NMDARe or healthy controls. NMDAR surface dynamics were assessed with single-molecule imaging by photoactivated localization microscopy. RESULTS: NMDAR trajectories from neurons treated with SGE-301 were less confinement, with increased diffusion coefficients. This effect mainly occurred at synapses because extrasynaptic diffusion and confinement were minimally affected by SGE-301. Treatment with patients' IgG reduced NMDAR surface dynamics and increased their confinement. Remarkably, SGE-301 incubation antagonized patients' IgG effects in both synaptic and extrasynaptic membrane compartments, restoring diffusion and confinement values similar to those from neurons exposed to control IgG. DISCUSSION: We demonstrate that SGE-301 upregulates NMDAR surface diffusion and antagonizes the pathogenic effects of patients' IgG on NMDAR membrane organization. These findings suggest a potential therapeutic strategy for NMDARe.
Assuntos
Encefalite Antirreceptor de N-Metil-D-Aspartato , Hipocampo , Imunoglobulina G , Neurônios , Receptores de N-Metil-D-Aspartato , Humanos , Hipocampo/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/imunologia , Animais , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Imunoglobulina G/farmacologia , Regulação Alostérica/efeitos dos fármacos , Células Cultivadas , Autoanticorpos/farmacologia , Feminino , Masculino , Ratos , Adulto , Imagem Individual de MoléculaRESUMO
INTRODUCTION: Implant infections caused by Staphylococcus aureus are responsible for high mortality and morbidity worldwide. Treatment of these infections can be difficult especially when bacterial biofilms are involved. In this study we investigate the potential of infrared photoimmunotherapy to eradicate staphylococcal infection in a mouse model. METHODS: A monoclonal antibody that targets Wall Teichoic Acid surface components of both S. aureus and its biofilm (4497-IgG1) was conjugated to a photosensitizer (IRDye700DX) and used as photoimmunotherapy in vitro and in vivo in mice with a subcutaneous implant pre-colonized with biofilm of Staphylococcus aureus. A dose of 400 µg and 200 µg of antibody-photosensitizer conjugate 4497-IgG-IRDye700DXwas administered intravenously to two groups of 5 mice. In addition, multiple control groups (vancomycin treated, unconjugated IRDye700DX and IRDye700DX conjugated to a non-specific antibody) were used to verify anti-microbial effects. RESULTS: In vitro results of 4497-IgG-IRDye700DX on pre-colonized (biofilm) implants showed significant (p<0.01) colony-forming units (CFU) reduction at a concentration of 5 µg of the antibody-photosensitizer conjugate. In vivo, treatment with 4497-IgG-IRDye700DX showed no significant CFU reduction at the implant infection. However, tissue around the implant did show a significant CFU reduction with 400 µg 4497-IgG-IRDye700DX compared to control groups (p = 0.037). CONCLUSION: This study demonstrated the antimicrobial potential of photoimmunotherapy for selectively eliminating S. aureus in vivo. However, using a solid implant instead of a catheter could result in an increased bactericidal effect of 4497-IgG-IRDye700DX and administration locally around an implant (per operative) could become valuable applications in patients that are difficult to treat with conventional methods. We conclude that photoimmunotherapy could be a potential additional therapy in the treatment of implant related infections, but requires further improvement.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Animais , Camundongos , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes , Imunoglobulina G/farmacologiaRESUMO
Immunoglobulin G (IgG) antibodies in the form of high-dose intravenous immunoglobulin (IVIG) exert immunomodulatory activity and are used in this capacity to treat inflammatory and autoimmune diseases. Reductionist approaches have revealed that terminal sialylation of the single asparagine-linked (N-linked) glycan at position 297 of the IgG1 Fc bestows antiinflammatory activity, which can be recapitulated by introduction of an F241A point mutation in the IgG1 Fc (FcF241A). Here, we examined the antiinflammatory activity of CHO-K1 cell-produced FcF241A in vivo in models of autoimmune inflammation and found it to be independent of sialylation. Intriguingly, sialylation markedly improved the half-life and bioavailability of FcF241A via impaired interaction with the asialoglycoprotein receptor ASGPR. Further, FcF241A suppressed inflammation through the same molecular pathways as IVIG and sialylated IgG1 Fc and required the C-type lectin SIGN-R1 in vivo. This contrasted with FcAbdeg (efgartigimod), an engineered IgG1 Fc with enhanced neonatal Fc receptor (FcRn) binding, which reduced total serum IgG concentrations, independent of SIGN-R1. When coadministered, FcF241A and FcAbdeg exhibited combinatorial antiinflammatory activity. Together, these results demonstrated that the antiinflammatory activity of FcF241A requires SIGN-R1, similarly to that of high-dose IVIG and sialylated IgG1, and can be used in combination with other antiinflammatory therapeutics that rely on divergent pathways, including FcAbdeg.
Assuntos
Imunoglobulina G , Imunoglobulinas Intravenosas , Recém-Nascido , Humanos , Imunoglobulina G/genética , Imunoglobulina G/farmacologia , Imunoglobulinas Intravenosas/uso terapêutico , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/farmacologia , Inflamação/genética , Inflamação/tratamento farmacológico , Receptores Fc/genética , GlicosilaçãoRESUMO
BACKGROUND: The safety of immune-checkpoint inhibitors (ICIs) has not been thoroughly investigated in non-small cell lung cancer (NSCLC) patients with chronic hepatitis B (CHB) or occult hepatitis B infection (OBI). The authors analyzed the incidence of hepatitis B virus (HBV) reactivation, immune-related hepatitis and jaundice in NSCLC patients in a real-world setting. METHODS: A total of 1277 NSCLC patients treated with ICIs were analyzed. Among them, 52 patients were hepatitis B surface antigen (HBsAg) (+) (group A, CHB), 759 patients were HBsAg (-)/hepatitis B core antibody immunoglobulin G (anti-HBc IgG) (+) (group B, OBI), and 466 patients were HBsAg (-)/anti-HBc IgG (-) (group C). Among the 52 patients with CHB, 38 (73.1%) were receiving antiviral therapy. The primary end point was HBV reactivation, immune-related hepatitis, and jaundice. The secondary end points included other immune-related adverse events and efficacy. RESULTS: HBV reactivation was observed in two patients (0.2%) who were both in group A (CHB). Among CHB patients who were not receiving antiviral therapy, HBV reactivation was observed in 14.3% (2 of 14 patients). The incidences of immune-related hepatitis and jaundice were comparable among the three groups. The incidence of ≥grade 3 other immune-related adverse events and efficacy were all comparable among the three groups (p > .05 for all comparisons). CONCLUSIONS: In this large, real-world cohort study, the safety and efficacy of ICIs were comparable in patients with CHB and OBI. HBV reactivation was observed in patients with CHB without antiviral therapy indicating antiviral prophylaxis should be required for them. For patients with OBI, the risk of HBV reactivation was minimal.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Hepatite B Crônica , Hepatite B , Icterícia , Neoplasias Pulmonares , Humanos , Vírus da Hepatite B , Inibidores de Checkpoint Imunológico/efeitos adversos , Hepatite B Crônica/complicações , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/epidemiologia , Antígenos de Superfície da Hepatite B/farmacologia , Antígenos de Superfície da Hepatite B/uso terapêutico , Incidência , Carcinoma Pulmonar de Células não Pequenas/complicações , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/epidemiologia , Estudos de Coortes , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/induzido quimicamente , Antivirais/efeitos adversos , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Icterícia/induzido quimicamente , Icterícia/complicações , Icterícia/tratamento farmacológico , Hepatite B/complicações , Ativação Viral , DNA ViralRESUMO
AIMS: To identify an effective strategy for promoting microvascular endothelial cells (MECs) to phagocytize myelin debris and reduce secretion of inflammatory factors following spinal cord injury (SCI). METHODS: We established a coculture model of myelin debris and vascular-like structures. The efficiency with which MECs phagocytize myelin debris under different conditions was examined via ELISA, flow cytometry, and immunofluorescence. Tubastatin-A was used to interfere with the coculture model. The anti-inflammatory effects of Tubastatin-A were observed by HE staining, flow cytometry, immunofluorescence, and ELISA. RESULTS: MECs phagocytized myelin debris via IgM opsonization, and phagocytosis promoted the secretion of inflammatory factors, whereas IgG-opsonized myelin debris had no effect on inflammatory factors. Application of the HDAC6 inhibitor Tubastatin-A increased the IgG levels and decreased the IgM levels by regulating the proliferation and differentiation of B cells. Tubastatin-A exerted a regulatory effect on the HDAC6-mediated autophagy-lysosome pathway, promoting MECs to phagocytize myelin debris, reducing the secretion of inflammatory factors, and accelerating the repair of SCI. CONCLUSIONS: Inhibition of HDAC6 to regulate the immune-inflammatory response and promote MECs to phagocytize myelin debris may represent a novel strategy in the treatment of SCI.
Assuntos
Bainha de Mielina , Traumatismos da Medula Espinal , Humanos , Células Endoteliais/metabolismo , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/metabolismo , Imunoglobulina G/farmacologia , Imunoglobulina M/metabolismo , Macrófagos , Bainha de Mielina/metabolismo , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismoRESUMO
Objective: To explore the expression of endosialin, i.e., CD248 in human hypertrophic scars (HSs) and its regulatory effect on the phenotype of hypertrophic scar fibroblasts (HSFs). Methods: The method of experimental research was used. From March to May, 2023, 3 pediatric patients with HS were admitted to the Department of Burns and Cutaneous Surgery of the First Affiliated Hospital of Air Force Medical University, including 2 females and 1 male, aged one year ten months to two years. The HS tissue resected during the surgery and the remaining full-thickness skin graft, i.e., normal skin tissue after full-thickness skin grafting were collected from the aforementioned pediatric patients for subsequent experiments. Using the aforementioned two types of tissue, the histological structures were observed by hematoxylin-eosin staining, collagen distribution was observed by Masson staining, and the expression of CD248 was observed and measured by immunohistochemical staining. The primary HSFs were isolated from HS tissue using explant culture technique, and the 3rd to 5th passages of HSFs were used in subsequent experiments. According to the random number table, HSFs were divided into immunoglobulin G78 (IgG78)-treated group and IgG control group, which were treated with 200 nmol/L human CD248 monoclonal antibody IgG78 and human IgG control antibody for 24 h, respectively. The mRNA expressions of collagen type â (Col â ) and α-smooth muscle actin (α-SMA) in HSFs were measured by real-time fluorescence quantitative reverse transcription polymerase chain reaction, the protein expressions of Col â and α-SMA in HSFs were detected by Western blotting, and the intracellular location and protein expressions of Col â and α-SMA were detected by immunofluorescence method. The number of samples in each experiment was 3. Data were statistically analyzed with paired sample t test and independent sample t test. Results: Compared with those in normal skin tissue, the epidermis and dermis in HS tissue were significantly thicker, with massive accumulation and disordered arrangement of collagen in the dermis. The expression of CD248 in HS tissue was significantly upregulated compared with that in normal skin tissue (t=5.29, P<0.05). At post treatment hour 24, the mRNA expressions of Col â and α-SMA of HSFs in IgG78-treated group were 0.39±0.05 and 0.56±0.09, respectively, which were significantly lower than 1.00±0.07 and 1.00±0.08 in IgG control group, respectively (with t values of 11.87 and 6.49, respectively, P values all <0.05). The protein expressions of Col â and α-SMA of HSFs in IgG78-treated group were 0.617±0.011 and 0.67±0.14, respectively, which were significantly lower than 1.259±0.052 and 1.23±0.16 in IgG control group, respectively (with t values of 20.92 and 4.52, respectively, P values all <0.05). At post treatment hour 24, immunofluorescence staining showed that Col â and α-SMA mainly located in the cytoplasm of HSFs in the two groups, and the protein expressions of Col â and α-SMA of HSFs in IgG78-treated group were obviously downregulated compared with those in IgG control group. Conclusions: The expression of CD248 is significantly upregulated in human HS. Targeted blockade of CD248 can significantly inhibit the collagen synthesis by HSFs and the transdifferentiation of HSFs into myofibroblasts.
Assuntos
Cicatriz Hipertrófica , Feminino , Humanos , Masculino , Criança , Cicatriz Hipertrófica/patologia , Fibroblastos/metabolismo , Colágeno/metabolismo , RNA Mensageiro/genética , Fenótipo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/farmacologia , Antígenos CD/metabolismo , Antígenos CD/farmacologiaRESUMO
Maternal immune activation results in altered levels of cytokines in the maternal-fetal system, which has a negative impact on fetal development, including the gonadotropin-releasing hormone (GnRH) system, which is crucial for the reproduction. Suppression of GnRH-neuron migration may be associated with cytokine imbalances, and primarily with proinflammatory cytokine interleukin (IL)-6. This study aimed to determine the effects of IL-6 and monoclonal antibody to IL-6 or IL-6R or polyclonal IgG on the formation of migration route of GnRH-neurons in ex vivo and in vivo rodent models on day 11.5 of embryonic development. The increased level of IL-6 in mouse nasal explants suppressed peripherin-positive fiber outgrowth, while this led to an increase in the number of GnRH-neurons in the nose and olfactory bulbs and a decrease in their number in the fetal brain. This effect is likely to be realized via IL-6 receptors along the olfactory nerves. The suppressive effect of IL-6 was diminished by monoclonal antibodies to IL-6 or its receptors and by IgG.
Assuntos
Citocinas , Hormônio Liberador de Gonadotropina , Animais , Feminino , Camundongos , Gravidez , Encéfalo/metabolismo , Movimento Celular , Citocinas/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Imunoglobulina G/farmacologia , Interleucina-6/farmacologia , Roedores/metabolismoRESUMO
Exposure to mercury (Hg) and silver (Ag) has been shown to induce autoimmune diseases in genetically susceptible rodents. Here, A.SW mice were initially exposed to HgCl2, AgNO3 or tap water (control) for 3 weeks. After 13 weeks of stoppage, all mice had secondary exposure to 203HgCl2. After secondary exposure, higher and earlier ANoA titers were observed in mice initially exposed to Hg or Ag compared to control. Further, mice initially exposed to Ag showed higher total IgG1 and IgG2a, Whole Body Retention and lymph nodes and spleen accumulation of Hg compared to mice initially exposed to Hg and controls. These findings showed an earlier and stronger immunological response in A.SW mice compared with control, following re-exposure to heavy metals indicating an immunological memory. Additionally, secondary exposure to a different heavy metal may aggravate the effects of exposure of at least one of the metals indicating cross-reactivity.
Assuntos
Doenças Autoimunes , Mercúrio , Camundongos , Animais , Autoimunidade , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/genética , Predisposição Genética para Doença , Imunoglobulina G/farmacologia , AceleraçãoRESUMO
Previously, we and others have shown that SARS-CoV-2 spike-specific IgG antibodies play a major role in disease severity in COVID-19 by triggering macrophage hyperactivation, disrupting endothelial barrier integrity, and inducing thrombus formation. This hyperinflammation is dependent on high levels of anti-spike IgG with aberrant Fc tail glycosylation, leading to Fcγ receptor hyperactivation. For development of immune-regulatory therapeutics, drug specificity is crucial to counteract excessive inflammation whereas simultaneously minimizing the inhibition of antiviral immunity. We here developed an in vitro activation assay to screen for small molecule drugs that specifically counteract antibody-induced pathology. We identified that anti-spike-induced inflammation is specifically blocked by small molecule inhibitors against SYK and PI3K. We identified SYK inhibitor entospletinib as the most promising candidate drug, which also counteracted anti-spike-induced endothelial dysfunction and thrombus formation. Moreover, entospletinib blocked inflammation by different SARS-CoV-2 variants of concern. Combined, these data identify entospletinib as a promising treatment for severe COVID-19.
Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Anticorpos Antivirais , Inflamação/tratamento farmacológico , Imunoglobulina G/farmacologiaRESUMO
Acute lung injury is significantly associated with the aberrant activation and pyroptosis of alveolar macrophages. Targeting the GPR18 receptor presents a potential therapeutic approach to mitigate inflammation. Verbenalin, a prominent component of Verbena in Xuanfeibaidu (XFBD) granules, is recommended for treating COVID-19. In this study, we demonstrate the therapeutic effect of verbenalin on lung injury through direct binding to the GPR18 receptor. Verbenalin inhibits the activation of inflammatory signaling pathways induced by lipopolysaccharide (LPS) and IgG immune complex (IgG IC) via GPR18 receptor activation. The structural basis for verbenalin's effect on GPR18 activation is elucidated through molecular docking and molecular dynamics simulations. Furthermore, we establish that IgG IC induces macrophage pyroptosis by upregulating the expression of GSDME and GSDMD through CEBP-δ activation, while verbenalin inhibits this process. Additionally, we provide the first evidence that IgG IC promotes the formation of neutrophil extracellular traps (NETs), and verbenalin suppresses NETs formation. Collectively, our findings indicate that verbenalin functions as a "phytoresolvin" to promote inflammation regression and suggests that targeting the C/EBP-δ/GSDMD/GSDME axis to inhibit macrophage pyroptosis may represent a novel strategy for treating acute lung injury and sepsis.
Assuntos
Lesão Pulmonar Aguda , COVID-19 , Sepse , Humanos , Complexo Antígeno-Anticorpo/efeitos adversos , Simulação de Acoplamento Molecular , Lesão Pulmonar Aguda/tratamento farmacológico , Sepse/tratamento farmacológico , Inflamação , Imunoglobulina G/farmacologia , Receptores Acoplados a Proteínas GRESUMO
The present study was performed to investigate the effects of dietary supplementation with herbal additives on meat quality, slaughter performance and the cecal microbial community in Hungarian white geese. A total of 60 newborn geese were assigned equally into the control group (CON) and the herbal complex supplemented group (HS). The dietary supplementations consisted of Compound Herbal Additive A (CHAA) including Pulsatilla, Gentian and Rhizoma coptidis, and Compound Herbal Additive B (CHAB) containing Codonopsis pilosula, Atractylodes, Poria cocos and Licorice. The geese in the HS group received a basal diet supplemented with 0.2% CHAA from day 0 to day 42 at the postnatal stage. Then from day 43 to day 70, the geese in HS group were provide a basal diet with 0.15% CHAB. The geese in the CON group were only provided with the basal diet. The results showed that the slaughter rate (SR), half chamber rates (HCR), eviscerated rate (ER) and breast muscle rate (BMR) in the HS group tended to increase slightly compared with the CON group (ns). In addition, the shear force, filtration rate and pH value of breast muscle and thigh muscle in the HS group were slightly enhanced compared to the CON group (ns). Significant increased levels in carbohydrate content, fat content and energy (P < 0.01) and significant decreased levels in cholesterol content (P < 0.01) were observed in the muscle of the HS group. The total amino acid (Glu, Lys, Thr and Asp) content in the muscle increased in HS group than in the CON group (P < 0.01). Dietary herb supplementations significantly increased the levels of IgG in serum (P < 0.05) on day 43 and higher levels of IgM, IgA and IgG (P < 0.01) were also observed in the HS group on day 70. Furthermore, 16S rRNA sequencing results indicated that herbal additives increased the growth of beneficial bacteria and inhibited the proliferation of harmful bacteria in the geese caecum. Altogether, these results offer crucial insights into the potential benefits of incorporating CHAA and CHAB into the diets of Hungarian white goose. The findings indicate that such supplementations could significantly improve meat quality, regulate the immune system and shape the intestinal microbiota composition.
Assuntos
Microbioma Gastrointestinal , Gansos , Animais , Humanos , Recém-Nascido , Hungria , RNA Ribossômico 16S , Suplementos Nutricionais/análise , Músculo Esquelético , Carne/análise , Ceco , Imunoglobulina G/farmacologiaRESUMO
The primary objectives of this research were to investigate the protective effects of liriodendrin against IgG immune complex (IgG-IC)-induced acute lung injury (ALI) and to elucidate the underlying mechanisms. This study employed a mouse and cell model of IgG-IC-induced acute lung injury. Lung tissue was stained with hematoxylin-eosin to observe pathological alterations and arterial blood gas analysis was tested. Inflammatory cytokines, including interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-alpha (TNF-α), were measured using ELISA. The mRNA expression of inflammatory cytokines was assessed via RT-qPCR. Molecular docking and enrichment analysis were combined to identify the most potential signaling pathways modulated by liriodendrin, which were then verified using western blot analysis in IgG-IC-induced ALI models. We identified 253 shared targets between liriodendrin and IgG-IC-induced acute lung injury from the database. Through network pharmacology, enrichment analysis, and molecular docking, SRC was determined to be the most closely associated target of liriodendrin in IgG-IC-induced ALI. Pretreatment with liriodendrin notably reduced the increased cytokine secretion of IL-1ß, IL-6, and TNF-α. Histopathological analysis of lung tissue demonstrated a protective effect of liriodendrin on IgG-IC-induced acute lung injury in mice. Arterial blood gas analysis showed liriodendrin ameliorated acidosis and hypoxemia efficiently. Further studies revealed that liriodendrin pretreatment substantially attenuated the elevated phosphorylation levels of SRC's downstream components (JNK, P38, and STAT3), suggesting that liriodendrin may protect against IgG-IC-induced ALI via the SRC/STAT3/MAPK pathway. Our findings indicate that liriodendrin protects against IgG-IC-induced acute lung injury by inhibiting the SRC/STAT3/MAPK signaling pathway, suggesting that liriodendrin may serve as a potential treatment for acute lung injury caused by IgG-IC.
Assuntos
Lesão Pulmonar Aguda , Complexo Antígeno-Anticorpo , Camundongos , Animais , Complexo Antígeno-Anticorpo/farmacologia , Complexo Antígeno-Anticorpo/uso terapêutico , Interleucina-6 , Fator de Necrose Tumoral alfa/farmacologia , Simulação de Acoplamento Molecular , Farmacologia em Rede , Transdução de Sinais , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/prevenção & controle , Lesão Pulmonar Aguda/etiologia , Pulmão/patologia , Citocinas/metabolismo , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Lipopolissacarídeos/farmacologiaRESUMO
This study aimed to clarify the effects of two processed forms of American ginseng (Panax quinquefolius L.) on immunosuppression caused by cyclophosphamide (CTX) in mice. In the CTX-induced immunosuppressive model, mice were given either steamed American ginseng (American ginseng red, AGR) or raw American ginseng (American ginseng soft branch, AGS) by intragastric administration. Serum and spleen tissues were collected, and the pathological changes in mice spleens were observed by conventional HE staining. The expression levels of cytokines were detected by ELISA, and the apoptosis of splenic cells was determined by western blotting. The results showed that AGR and AGS could relieve CTX-induced immunosuppression through the enhanced immune organ index, improved cell-mediated immune response, increased serum levels of cytokines (TNF-α, IFN-γ, and IL-2) and immunoglobulins (IgG, IgA, and IgM), as well as macrophage activities including carbon clearance and phagocytic index. AGR and AGS downregulated the expression of BAX and elevated the expression of Bcl-2, p-P38, p-JNK, and p-ERK in the spleens of CTX-injected animals. Compared to AGS, AGR significantly improved the number of CD4+CD8-T lymphocytes, the spleen index, and serum levels of IgA, IgG, TNF-α, and IFN-γ. The expression of the ERK/MAPK pathway was markedly increased. These findings support the hypothesis that AGR and AGS are effective immunomodulatory agents capable of preventing immune system hypofunction. Future research may investigate the exact mechanism to rule out any unforeseen effects of AGR and AGS.
Assuntos
Panax , Fator de Necrose Tumoral alfa , Camundongos , Animais , Fator de Necrose Tumoral alfa/farmacologia , Ciclofosfamida/efeitos adversos , Terapia de Imunossupressão , Citocinas/metabolismo , Macrófagos , Imunoglobulina G/farmacologia , Transdução de Sinais , Imunoglobulina A/farmacologiaRESUMO
AIM: Enhanced cardiac sympathetic afferent reflex (CSAR) promotes sympathetic hyperactivation in chronic heart failure (CHF). Salusin-ß is a torsin family 2 member A (TOR2A) gene product and a cardiovascular active peptide closely associated with cardiovascular diseases. We aimed to determine the roles of salusin-ß in the paraventricular nucleus (PVN) in modulating enhanced CSAR and sympathetic hyperactivation in rats with CHF induced by coronary artery ligation and elucidate the underlying molecular mechanisms. METHODS: CSAR was evaluated based on the responses of mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) to the epicardial administration of capsaicin in rats under anesthesia. RESULTS: Salusin-ß protein expression was upregulated in the PVN of the CHF compared with sham-operated rats. Salusin-ß microinjection into the PVN dose-dependently increased MAP and RSNA and enhanced CSAR, while anti-salusin-ß IgG exerted opposite effects. The effect of salusin-ß was inhibited by reactive oxygen species (ROS) scavenger or NAD(P)H oxidase inhibitor but promoted by superoxide dismutase inhibitor. The effect of anti-salusin-ß IgG was interdicted by nitric oxide (NO) synthase inhibitor. Furthermore, chronic salusin-ß gene knockdown in PVN attenuated CSAR, reduced sympathetic output, improved myocardial remodeling and cardiac function, decreased NAD(P)H oxidase activity and ROS levels, and increased NO levels in the CHF rats. CONCLUSION: Increased salusin-ß activity in the PVN contributes to sympathetic hyperactivation and CSAR in CHF by inhibiting NO release and stimulating NAD(P)H oxidase-ROS production. Reducing endogenous central salusin-ß expression might be a novel strategy for preventing and treating CHF in the future.
Assuntos
Insuficiência Cardíaca , Núcleo Hipotalâmico Paraventricular , Ratos , Animais , Espécies Reativas de Oxigênio/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Vasos Coronários/metabolismo , Ratos Sprague-Dawley , Angiotensina II/farmacologia , Insuficiência Cardíaca/metabolismo , Reflexo/fisiologia , Sistema Nervoso Simpático , NADPH Oxidases/metabolismo , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Pressão Sanguínea/fisiologiaRESUMO
BACKGROUND: In â¼50% of severe atrioventricular blocks (AVBs) occurring in adults <50 years, the underlying etiology remains unknown. Preliminary evidence from case reports suggests that autoimmunity, specifically the presence of circulating anti-Ro/SSA antibodies in the patient (acquired form), in the patient's mother (late-progressive congenital form), or in both (mixed form), could be involved in a fraction of idiopathic AVBs in adults by possibly targeting the L-type calcium channel (Cav1.2) and inhibiting the related current (ICaL). OBJECTIVES: The purpose of this study was to evaluate whether anti-Ro/SSA antibodies are causally implicated in the development of isolated AVBs in adults. METHODS: Thirty-four consecutive patients with isolated AVB of unknown origin and 17 available mothers were prospectively enrolled in a cross-sectional study. Anti-Ro/SSA antibodies were assessed by fluoroenzyme-immunoassay, immuno-Western blotting, and line-blot immunoassay. Purified immunoglobulin-G (IgG) from anti-Ro/SSA-positive and anti-Ro/SSA-negative subjects were tested on ICaL and Cav1.2 expression using tSA201 and HEK293 cells, respectively. Moreover, in 13 AVB patients, the impact of a short course of steroid therapy on AV conduction was evaluated. RESULTS: Anti-Ro/SSA antibodies, particularly anti-Ro/SSA-52kD, were found in 53% of AVB-patients and/or in their mothers, most commonly an acquired or mixed form (two-thirds of cases) without history of autoimmune diseases. Purified IgG from anti-Ro/SSA-positive but not anti-Ro/SSA-negative AVB patients acutely inhibited ICaL and chronically down-regulated Cav1.2 expression. Moreover, anti-Ro/SSA-positive sera showed high reactivity with peptides corresponding to the Cav1.2 channel pore-forming region. Finally, steroid therapy rapidly improved AV conduction in AVB-patients with circulating anti-Ro/SSA antibodies but not in those without. CONCLUSIONS: Our study points to anti-Ro/SSA antibodies as a novel, epidemiologically relevant and potentially reversible cause of isolated AVB in adults, via an autoimmune-mediated functional interference with the L-type calcium channels. These findings have significant impact on antiarrhythmic therapies by avoiding or delaying pacemaker implantation.
Assuntos
Bloqueio Atrioventricular , Humanos , Adulto , Canais de Cálcio , Estudos Transversais , Células HEK293 , Imunoglobulina G/farmacologia , EsteroidesRESUMO
The digestive system is exposed to severe inflammation as a result of taking some medications that have gastrointestinal side effects. Sixty Swiss-albino male mice were randomly distributed into six groups to treat inflammations of the colon, stomach, and small intestine caused by taking high doses of diclofenac (D), with two novel synthesized compounds, pyrazolo [3,4 d] pyridazine derivatives (Co1 and Co2). Myeloperoxidase enzyme activity was determined in the colon and small intestinal tissues. Serum contents of TNF-α, IL-22, IgG, and IgM were determined by ELISA. Histopathological examinations of the colon, small intestinal, and stomach tissues were microscopically analyzed. TNF-α, IL-22, and TNFSF11 gene expression were measured in the colon, intestinal, and spleen using qRT-PCR. Diclofenac caused surface columnar epithelial cell loss, focal necrosis of the gastric mucosa, inflammatory cell infiltration, and congested blood vessels in the stomach, colon, and small intestinal tissues. Co1 component was found to be better than Co2 component in reducing the focal necrosis of gastric mucosa and improving the histological structures of the stomach, colon, and small intestinal tissues. After 14 days, the activity of the myeloperoxidase enzyme was increased in group D and decreased in groups DCo1, DCo2, Co1, and Co2. Serum concentrations of TNF-α and IgG were increased, while IL-22 and IGM were reduced in the D, DCo1, and DCo2 groups compared with the Co1 and control groups. TNF-α gene was upregulated in the D group and downregulated in the Co1 group, while the IL-22 gene was downregulated in the D group and upregulated in the Co1 group compared with the control group. The CO1 component may be useful in reducing digestive system inflammation.
Assuntos
Colite , Camundongos , Animais , Colite/tratamento farmacológico , Peroxidase/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Diclofenaco/farmacologia , Dióxido de Carbono/metabolismo , Dióxido de Carbono/farmacologia , Dióxido de Carbono/uso terapêutico , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Inflamação/patologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Colo , Antioxidantes/farmacologia , Necrose/tratamento farmacológico , Necrose/metabolismo , Necrose/patologia , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Imunoglobulina M/metabolismo , Imunoglobulina M/farmacologia , Imunoglobulina M/uso terapêutico , Modelos Animais de DoençasRESUMO
Polysorbates (PS) are nonionic surfactants that are commonly included in protein formulations to mitigate the formation of interfacial stress-induced protein particles and thus increase their long-term storage stability. Nonetheless, factors that dictate the efficiency of different polysorbates in mitigating protein particle formation, especially during the application of interfacial stresses, are often ill defined. Here, we used a Langmuir trough to determine the surface activity of two IgG1 monoclonal antibodies formulated with two different polysorbates (PS20 and PS80) when subjected to interfacial dilatational stress. Interfacial properties of these formulations were then correlated with characterization of subvisible protein particles measured by micro-flow imaging (MFI). Both mAbs, when formulated in PS20, demonstrate faster adsorption kinetics and higher surface activity compared to PS80 or surfactant-free formulations. Compression/expansion results suggest that when exposed to interfacial dilatational stresses, both mAb/PS20 formulations display interfacial properties of PS20 alone. In contrast, interfacial properties of both mAb/PS80 formulations suggest mAbs and PS80 are co-adsorbed to the air-water interface. Further, MFI analysis of the interface and the bulk solution confirms that PS20 is more effective than PS80 at mitigating the formation of larger particles in the bulk solution in both mAbs. Concomitantly, the efficiency of PS to prevent interface-induced protein particle formation also depended on the protein's inherent tendency to aggregate at a surfactant-free interface. Together, the studies presented here highlight the importance of determining the interfacial properties of mAbs, surfactants, and their combinations to make informed formulation decisions about the choice of surfactant.