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Tumor-associated neutrophil (TAN) effects on glioblastoma (GBM) biology remain under-characterized. We show here that neutrophils with dendritic features-including morphological complexity, expression of antigen presentation genes, and the ability to process exogenous peptide and stimulate major histocompatibility complex (MHC)II-dependent T cell activation-accumulate intratumorally and suppress tumor growth in vivo. Trajectory analysis of patient TAN scRNA-seq identifies this "hybrid" dendritic-neutrophil phenotype as a polarization state that is distinct from canonical cytotoxic TANs, and which differentiates from local precursors. These hybrid-inducible immature neutrophils-which we identified in patient and murine glioblastomas-arise not from circulation, but from local skull marrow. Through labeled skull flap transplantation and targeted ablation, we characterize calvarial marrow as a contributor of antitumoral myeloid antigen-presenting cells (APCs), including TANs, which elicit T cell cytotoxicity and memory. As such, agents augmenting neutrophil egress from skull marrow-such as intracalvarial AMD3100, whose survival-prolonging effect in GBM we report-present therapeutic potential.
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Neoplasias Encefálicas , Diferenciación Celular , Células Dendríticas , Glioblastoma , Neutrófilos , Humanos , Animales , Ratones , Neutrófilos/inmunología , Neutrófilos/metabolismo , Glioblastoma/patología , Glioblastoma/inmunología , Glioblastoma/genética , Glioblastoma/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Cráneo/patología , Cráneo/inmunología , Médula Ósea/patología , Médula Ósea/inmunología , Ratones Endogámicos C57BL , Línea Celular TumoralRESUMEN
Blockade of immune checkpoint receptors has shown outstanding efficacy for tumor immunotherapy. Promising treatment with anti-lymphocyte-activation gene-3 (LAG-3) has already been recognized as the next efficacious treatment, but there is still limited understanding of the mechanism of LAG-3-mediated immune suppression. Here, utilizing high-resolution molecular imaging, we find a mechanism of CD4 T cell suppression via LAG-3, in which LAG-3-bound major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) gather at the central region of an immunological synapse and are trans-endocytosed by T cell receptor-driven internalization motility toward CD4 and CD8 T cells expressing LAG-3. Downregulation of MHC class II molecules on APCs thus results in the attenuation of their antigen-presentation function and impairment of CD4 T cell activation. From these data, anti-LAG-3 treatment is suggested to have potency to directly block the inhibitory signaling via LAG-3 and simultaneously reduce MHC class II expression on APCs by LAG-3-mediated trans-endocytosis for recovery from T cell exhaustion.
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The first approved vaccines for human use against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are nanotechnology-based. Although they are modular, rapidly produced, and can reduce disease severity, the currently available vaccines are restricted in preventing infection, stressing the global demand for novel preventive vaccine technologies. Bearing this in mind, we set out to develop a flexible nanovaccine platform for nasal administration to induce mucosal immunity, which is fundamental for optimal protection against respiratory virus infection. The next-generation multiepitope nanovaccines co-deliver immunogenic peptides, selected by an immunoinformatic workflow, along with adjuvants and regulators of the PD-L1 expression. As a case study, we focused on SARS-CoV-2 peptides as relevant antigens to validate the approach. This platform can evoke both local and systemic cellular- and humoral-specific responses against SARS-CoV-2. This led to the secretion of immunoglobulin A (IgA), capable of neutralizing SARS-CoV-2, including variants of concern, following a heterologous immunization strategy. Considering the limitations of the required cold chain distribution for current nanotechnology-based vaccines, it is shown that the lyophilized nanovaccine is stable for long-term at room temperature and retains its in vivo efficacy upon reconstitution. This makes it particularly relevant for developing countries and offers a modular system adaptable to future viral threats.
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BACKGROUND & AIMS: Intestinal epithelial cell (IEC) damage is a hallmark of celiac disease (CeD); however, its role in gluten-dependent T-cell activation is unknown. We investigated IEC-gluten-T-cell interactions in organoid monolayers expressing human major histocompatibility complex class II (HLA-DQ2.5), which facilitates gluten antigen recognition by CD4+ T cells in CeD. METHODS: Epithelial major histocompatibility complex class II (MHCII) was determined in active and treated CeD, and in nonimmunized and gluten-immunized DR3-DQ2.5 transgenic mice, lacking mouse MHCII molecules. Organoid monolayers from DR3-DQ2.5 mice were treated with or without interferon (IFN)-γ, and MHCII expression was evaluated by flow cytometry. Organoid monolayers and CD4+ T-cell co-cultures were incubated with gluten, predigested, or not by elastase-producing Pseudomonas aeruginosa or its lasB mutant. T-cell function was assessed based on proliferation, expression of activation markers, and cytokine release in the co-culture supernatants. RESULTS: Patients with active CeD and gluten-immunized DR3-DQ2.5 mice demonstrated epithelial MHCII expression. Organoid monolayers derived from gluten-immunized DR3-DQ2.5 mice expressed MHCII, which was upregulated by IFN-γ. In organoid monolayer T-cell co-cultures, gluten increased the proliferation of CD4+ T cells, expression of T-cell activation markers, and the release of interleukin-2, IFN-γ, and interleukin-15 in co-culture supernatants. Gluten metabolized by P aeruginosa, but not the lasB mutant, enhanced CD4+ T-cell proliferation and activation. CONCLUSIONS: Gluten antigens are efficiently presented by MHCII-expressing IECs, resulting in the activation of gluten-specific CD4+ T cells, which is enhanced by gluten predigestion with microbial elastase. Therapeutics directed at IECs may offer a novel approach for modulating both adaptive and innate immunity in patients with CeD.
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Reducing the immunogenicity of animal-derived monoclonal antibodies (mAbs) for use in humans is critical to maximize therapeutic effectiveness and preclude potential adverse events. While traditional humanization methods have primarily focused on grafting antibody Complementarity-Determining Regions (CDRs) on homologous human antibody scaffolds, framework regions can also play essential roles in antigen binding. Here, we describe the humanization of the pan-HLA-DR mAb 44H10, a murine antibody displaying significant involvement of the framework region in antigen binding. Using a structure-guided approach, we identify and restore framework residues that directly interact with the antigen or indirectly modulate antigen binding by shaping the antibody paratope and engineer a humanized antibody with affinity, biophysical profile, and molecular binding basis comparable to that of the parental 44H10 mAb. As a humanized molecule, this antibody holds promise as a scaffold for the development of MHC class II-targeting therapeutics and vaccines.
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Avian influenza A virus (IAV) surveillance in Northern California, USA, revealed unique IAV hemagglutinin (HA) genome sequences in cloacal swabs from lesser scaups. We found two closely related HA sequences in the same duck species in 2010 and 2013. Phylogenetic analyses suggest that both sequences belong to the recently discovered H19 subtype, which thus far has remained uncharacterized. We demonstrate that H19 does not bind the canonical IAV receptor sialic acid (Sia). Instead, H19 binds to the major histocompatibility complex class II (MHC class II), which facilitates viral entry. Unlike the broad MHC class II specificity of H17 and H18 from bat IAV, H19 exhibits a species-specific MHC class II usage that suggests a limited host range and zoonotic potential. Using cell lines overexpressing MHC class II, we rescued recombinant H19 IAV. We solved the H19 crystal structure and identified residues within the putative Sia receptor binding site (RBS) that impede Sia-dependent entry.
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Patos , Glicoproteínas Hemaglutininas del Virus de la Influenza , Antígenos de Histocompatibilidad Clase II , Virus de la Influenza A , Filogenia , Receptores Virales , Animales , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Receptores Virales/metabolismo , Receptores Virales/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Patos/virología , Humanos , Internalización del Virus , Gripe Aviar/virología , Sitios de Unión , Unión Proteica , Cristalografía por Rayos X , Línea Celular , Ácido N-Acetilneuramínico/metabolismo , Especificidad del Huésped , Especificidad de la EspecieRESUMEN
BACKGROUND: Major histocompatibility complex (MHC) class II molecules expressed on B cells, monocytes and dendritic cells present processed peptides to CD4+ T cells as one of the mechanisms to combat infection and inflammation. AIM: To study MHC II expression in a variety of nonhuman primate species, including New World (NWM) squirrel monkeys (Saimiri boliviensis boliviensis), owl monkeys (Aotus nancymae), common marmosets (Callithrix spp.), and Old World (OWM) rhesus (Macaca mulatta), baboons (Papio anubis). METHODS: Two clones of cross-reactive mouse anti-human HLADR monoclonal antibodies (mAb) binding were analyzed by flow cytometry to evaluate MHC II expression on NHP immune cells, including T lymphocytes in whole blood (WB) and peripheral blood mononuclear cells (PBMC). RESULTS: MHC class II antibody reactivity is seen with CD20+ B cells, CD14+ monocytes and CD3+ T lymphocytes. Specific reactivity with both clones was demonstrated in T lymphocytes: this reactivity was not inhibited by purified CD16 antibody but was completely inhibited when pre-blocked with purified unconjugated MHC II antibody. Freshly prepared PBMC also showed reactivity with T lymphocytes without any stimulation. Interestingly, peripheral blood from rhesus macaques and olive baboons (OWM) showed no such T lymphocyte associated MHCII antibody reactivity. DISCUSSION & CONCLUSION: Our results from antibody (MHC II) reactivity clearly show the potential existence of constitutively expressed (with no stimulation) MHC II molecules on T lymphocytes in new world monkeys. These results suggest that additional study is warranted to evaluate the functional and evolutionary significance of these finding and to better understand MHC II expression on T lymphocytes in new world monkeys.
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Antígenos HLA-DR , Antígenos de Histocompatibilidad Clase II , Linfocitos T , Animales , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos HLA-DR/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Humanos , Macaca mulatta , Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Saimiri/inmunología , Callithrix/inmunología , Citometría de Flujo , Papio anubis/inmunología , Platirrinos/inmunologíaRESUMEN
BACKGROUND/AIMS: The major histocompatibility class II (MHC II) transactivator, known as CIITA, is induced by Interferon gamma (IFN-γ) and plays a well-established role in regulating the expression of class II MHC molecules in antigen-presenting cells. METHODS: Primary human hepatocytes (PHH) were isolated via therapeutic hepatectomy from two donors. The hepatocellular carcinoma (HCC) cell lines HepG2 and Huh7 were used for the mechanistic study, and HBV infection was performed in HepG2-NTCP cells. HBV DNA replication intermediates and secreted antigen levels were measured using Southern blotting and ELISA, respectively. RESULTS: We identified a non-canonical function of CIITA in the inhibition of hepatitis B virus (HBV) replication in both HCC cells and patient-derived PHH. Notably, in vivo experiments demonstrated that HBV DNA and secreted antigen levels were significantly decreased in mice injected with the CIITA construct. Mechanistically, CIITA inhibited HBV transcription and replication by suppressing the activity of HBV-specific enhancers/promoters. Indeed, CIITA exerts antiviral activity in hepatocytes through ERK1/2-mediated down-regulation of the expression of hepatocyte nuclear factor 1α (HNF1α) and HNF4α, which are essential factors for virus replication. In addition, silencing of CIITA significantly abolished the IFN-γ-mediated anti-HBV activity, suggesting that CIITA mediates the anti-HBV activity of IFN-γ to some extent. HBV X protein (HBx) counteracts the antiviral activity of CIITA via direct binding and impairing its function. CONCLUSION: Our findings reveal a novel antiviral mechanism of CIITA that involves the modulation of the ERK pathway to restrict HBV transcription. Additionally, our results suggest the possibility of a new immune avoidance mechanism involving HBx.
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Virus de la Hepatitis B , Hepatocitos , Proteínas Nucleares , Transactivadores , Replicación Viral , Virus de la Hepatitis B/fisiología , Humanos , Transactivadores/metabolismo , Transactivadores/genética , Animales , Ratones , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Hepatocitos/metabolismo , Hepatocitos/citología , Hepatocitos/virología , Células Hep G2 , Hepatitis B/metabolismo , Interferón gamma/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor Nuclear 1-alfa del Hepatocito/genética , ADN Viral/metabolismo , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Decreased monocytic HLA-DR expression is the most studied biomarker of immune competency in critically ill and autoimmune disease patients. However, the underlying regulatory mechanisms remain largely unknown. One probable HLA-DR dysregulation is through microRNAs. The aim of this study was to investigate the effects of specific microRNAs on HLA-DR expression in human monocytic cells. Four up- and four down-HLA-DR-regulating microRNAs were identified, with hsa-miR-let-7f-2-3p showing the most significant upregulation and hsa-miR-567 and hsa-miR-3972 downregulation. Anti-inflammatory glucocorticoid medication Dexamethasone-decreased HLA-DR was significantly restored by hsa-miR-let-7f-2-3p and hsa-miR-5693. Contrarily, proinflammatory cytokines IFN-γ and TNF-α-increased HLA-DR were significantly reversed by hsa-miR-567. Clinically, paired plasma samples from patients before and one day after cardiac surgery revealed up-regulated expression of hsa-miR-5693, hsa-miR-567, and hsa-miR-3972, following the major surgical trauma. In silico approaches were applied for functional microRNA-mRNA interaction prediction and candidate target genes were confirmed by qPCR analysis. In conclusion, novel monocytic HLA-DR microRNA modulators were identified and validated in vitro. Moreover, both the interaction between the microRNAs and anti- and proinflammatory molecules and the up-regulated microRNAs identified in cardiac surgery highlight the potential clinical relevance of our findings.
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Antígenos HLA-DR , MicroARNs , Monocitos , Humanos , MicroARNs/genética , Monocitos/inmunología , Monocitos/metabolismo , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Antígenos HLA-DR/metabolismo , Regulación de la Expresión Génica , Masculino , Factor de Necrosis Tumoral alfa/metabolismo , Femenino , Dexametasona/farmacologíaRESUMEN
Background and Objectives: Chlamydia trachomatis (C. trachomatis) represents one of the most prevalent bacterial sexually transmitted diseases. This study aims to explore the relationship between HLA alleles/genotypes/haplotypes and C. trachomatis infection to better understand high-risk individuals and potential complications. Materials and Methods: This prospective study recruited participants from Transylvania, Romania. Patients with positive NAAT tests for C. trachomatis from cervical/urethral secretion or urine were compared with controls regarding HLA-DR and -DQ alleles. DNA extraction for HLA typing was performed using venous blood samples. Results: Our analysis revealed that the presence of the DRB1*13 allele significantly heightened the likelihood of C. trachomatis infection (p = 0.017). Additionally, we observed that individuals carrying the DRB1*01/DRB1*13 and DQB1*03/DQB1*06 genotype had increased odds of C. trachomatis infection. Upon adjustment, the association between the DRB1*01/DRB1*13 genotype and C. trachomatis remained statistically significant. Conclusions: Our findings underscore the importance of specific HLA alleles and genotypes in influencing susceptibility to C. trachomatis infection. These results highlight the intricate relationship between host genetics and disease susceptibility, offering valuable insights for targeted prevention efforts and personalized healthcare strategies.
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Infecciones por Chlamydia , Chlamydia trachomatis , Polimorfismo Genético , Enfermedades de Transmisión Sexual , Adolescente , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Alelos , Infecciones por Chlamydia/genética , Predisposición Genética a la Enfermedad , Genotipo , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Estudios Prospectivos , Rumanía , Enfermedades de Transmisión Sexual/genéticaRESUMEN
Study of CD4+ T cell response and T cell receptor (TCR) specificity is crucial for understanding etiology of immune-mediated diseases and developing targeted therapies. However, solubility, accessibility, and stability of synthetic antigenic peptides used in T cell assays may be a critical point in such studies. Here we present a T cell activation reporter system using recombinant proteins containing antigenic epitopes fused with bacterial thioredoxin (trx-peptides) and obtained by bacterial expression. We report that co-incubation of CD4+ HA1.7 TCR+ reporter Jurkat 76 TRP cells with CD80+ HLA-DRB1*01:01+ HeLa cells or CD4+ Ob.1A12 TCR+ Jurkat 76 TRP with CD80+ HLA-DRB1*15:01+ HeLa cells resulted in activation of reporter Jurkat 76 TPR after addition of recombinant trx-peptide fusion proteins, containing TCR-specific epitopes. Trx-peptides were comparable with corresponding synthetic peptides in their capacity to activate Jurkat 76 TPR. These data demonstrate that thioredoxin as a carrier protein (trx) for antigenic peptides exhibits minimal interference with recognition of MHC-specific peptides by TCRs and consequent T cell activation. Our findings highlight potential feasibility of trx-peptides as a reagent for assessing the immunogenicity of antigenic fragments.
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Linfocitos T CD4-Positivos , Péptidos , Receptores de Antígenos de Linfocitos T , Proteínas Recombinantes de Fusión , Tiorredoxinas , Humanos , Tiorredoxinas/inmunología , Tiorredoxinas/genética , Células Jurkat , Linfocitos T CD4-Positivos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Péptidos/farmacología , Péptidos/inmunología , Péptidos/química , Activación de Linfocitos/efectos de los fármacos , Células HeLaRESUMEN
Background: Parkinson's disease (PD) is characterized by alpha-synuclein (α-Syn) pathology, neurodegeneration and neuroinflammation. Human leukocyte antigen (HLA) variants associated with PD and α-Syn specific CD4+ T lymphocytes in PD patients highlight the importance of antigen presentation in PD etiology. The class II transactivator (CIITA) regulates major histocompatibility complex class II (MHCII) expression. Reduced Ciita levels significantly increase α-Syn pathology, nigrostriatal neurodegeneration and behavioral deficits in α-Syn-induced rat PD models. Objective: Characterize immune profiles associated with enhanced PD-like pathology observed in rats expressing lower Ciita levels (DA.VRA4) compared to the background strain (DA). Methods: To model PD, we combined rAAV-mediated α-Syn overexpression in the substantia nigra with striatal injection of α-Syn preformed fibrils. Immune profiles in brain and blood were analyzed by flow cytometry and multiplexed ELISA in naïve rats, 4- and 8 weeks post rAAV injection. Results: Flow cytometry showed Ciita-dependent regulation of MHCII on microglia, brain macrophages and circulating myeloid cells. The MHCII-dependent microglial response was highest at 4 weeks post rAAV injection, whereas the MHCII levels in circulating myeloid cells was highest at 8 weeks. There was no major infiltration of macrophages or T lymphocytes into the CNS in response to α-Syn and only subtle Ciita- and/or α-Syn-dependent changes in the T lymphocyte compartment. Lower Ciita levels were consistently associated with higher TNF levels in serum. Conclusions: Ciita regulates susceptibility to PD-like pathology through minor but detectable changes in resident and peripheral immune cells and TNF levels, indicating that mild immunomodulatory therapies could have therapeutic effects in PD.
Parkinson's disease is characterized by loss of nerve cells. There is also abnormal aggregation of a protein called alpha-synuclein and an ongoing inflammatory response. Findings that immune cells in the blood of individuals with Parkinson's disease react against the alpha-synuclein protein and that genes important for the immune system affect the risk of developing Parkinson's disease indicate that immune responses are important in Parkinson's disease. We have previously found that a low expression of certain immune molecules worsens disease progression in a rat model of Parkinson's disease. The aim of this study was to identify changes in the immune system in rats that are associated with disease severity, to identify mechanisms that could be targeted to treat Parkinson's disease. To model Parkinson's disease, we injected a modified virus to produce large amounts of alpha-synuclein combined with an injection of aggregated alpha-synuclein proteins in the rat brain. The model mimics several features of Parkinson's disease including nerve cell death, problems with movement, accumulation of alpha-synuclein in the brain, and an immune response. We observed that the immune system in the brain and blood responded to the model but that differences were small compared to controls. Our results suggest that small changes in the immune system can have a large effect on disease progression and that therapies targeting the immune system are worth exploring to find better treatment for Parkinson's disease.
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Modelos Animales de Enfermedad , Enfermedad de Parkinson , Transactivadores , alfa-Sinucleína , Animales , alfa-Sinucleína/metabolismo , Ratas , Transactivadores/genética , Enfermedad de Parkinson/inmunología , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/terapia , Proteínas Nucleares/metabolismo , Sustancia Negra/patología , Sustancia Negra/metabolismo , Sustancia Negra/inmunología , Masculino , Dependovirus , Microglía/inmunología , Microglía/metabolismo , Microglía/patologíaRESUMEN
Transgenic mice expressing human major histocompatibility complex class II (MHCII) risk alleles are widely used in autoimmune disease research, but limitations arise due to non-physiologic expression. To address this, physiologically relevant mouse models are established via knock-in technology to explore the role of MHCII in diseases like rheumatoid arthritis. The gene sequences encoding the ectodomains are replaced with the human DRB1*04:01 and 04:02 alleles, DRA, and CD74 (invariant chain) in C57BL/6N mice. The collagen type II (Col2a1) gene is modified to mimic human COL2. Importantly, DRB1*04:01 knock-in mice display physiologic expression of human MHCII also on thymic epithelial cells, in contrast to DRB1*04:01 transgenic mice. Humanization of the invariant chain enhances MHCII expression on thymic epithelial cells, increases mature B cell numbers in spleen, and improves antigen presentation. To validate its functionality, the collagen-induced arthritis (CIA) model is used, where DRB1*04:01 expression led to a higher susceptibility to arthritis, as compared with mice expressing DRB1*04:02. In addition, the humanized T cell epitope on COL2 allows autoreactive T cell-mediated arthritis development. In conclusion, the humanized knock-in mouse faithfully expresses MHCII, confirming the DRB1*04:01 alleles role in rheumatoid arthritis and being also useful for studying MHCII-associated diseases.
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Alelos , Antígenos de Diferenciación de Linfocitos B , Artritis Reumatoide , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Antígenos de Histocompatibilidad Clase II , Ratones Endogámicos C57BL , Ratones Transgénicos , Animales , Ratones , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/inmunología , Humanos , Técnicas de Sustitución del Gen/métodos , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Artritis Experimental/genética , Artritis Experimental/inmunología , Cadenas HLA-DRB1/genética , Cadenas HLA-DRB1/inmunología , Colágeno Tipo II/genética , Colágeno Tipo II/inmunologíaRESUMEN
Efficient delivery of a DNA plasmid into antigen-presenting cells (APCs) is a potential strategy to enhance the immune responses of DNA vaccines. The bacterial ghost (BG) is a potent DNA vaccine delivery system that targets APCs. In the present work, we describe a new strategy of using E. coli BGs as carriers for an Ii-linked Hepatitis C Virus (HCV) NS3 DNA vaccine that improved both the transgene expression level and the antigen-presentation level in APCs. BGs were prepared from DH5α cells, characterized via electron microscopy and loaded with the DNA vaccine. The high transfection efficiency mediated using BGs was first evaluated in vitro, and then, the immune protective effect of the BG-Ii-NS3 vaccine was determined in vivo. It was found that the antibody titer in the sera of BG-Ii-NS3-challenged mice was higher than that of Ii-NS3-treated mice, indicating that the BGs enhanced the humoral immune activity of Ii-NS3. The cellular immune protective effect of the BG-Ii-NS3 vaccine was determined using long-term HCV NS3 expression in a mouse model in which luciferase was used as a reporter for HCV NS3 expression. Our results showed that the luciferase activity in BG-Ii-NS3-treated mice was significantly reduced compared with that in Ii-NS3-treated mice. The CTL assay results demonstrated that BG-Ii-NS3 induced a greater NS3-specific T-cell response than did Ii-NS3. In summary, our study demonstrated that BGs enhanced both the humoral and cellular immune response to the Ii-NS3 DNA vaccine and improved its immune protection against HCV infection.
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Major histocompatibility complex class II (MHC-II) deficiency or bare lymphocyte syndrome (BLS) is a rare, early-onset, autosomal recessive, and life-threatening inborn error of immunity. We aimed to assess the demographic, clinical, laboratory, follow-up, and treatment characteristics of patients with MHC-II deficiency, together with their survival. We retrospectively investigated 21 patients with MHC-II deficiency. Female/male ratio was 1.63. The median age at diagnosis was 16.3 months (5 months-9.7 years). Nineteen patients (90.5%) had parental consanguinity. Pulmonary diseases (pneumonia, chronic lung disease) (81%), diarrhoea (47.6%), and candidiasis (28.6%) were common. Four (19%) had autoimmunity, two developed septic arthritis, and three (14%) developed bronchiectasis in the follow-up. Three patients (14%) had CMV viraemia, one with bilateral CMV retinitis. Eight (38.1%) had lymphocytopenia, and four (19%) had neutropenia. Serum IgM, IgA, and IgG levels were low in 18 (85.7%), 15 (71.4%), and 11 (52.4%) patients, respectively. CD4+ lymphocytopenia, a reversed CD4+/CD8+ ratio, and absent/low HLA-DR expressions were detected in 93.3%, 86.7%, and 100% of the patients, respectively. Haematopoietic stem cell transplantation (HSCT) was performed on nine patients, and four died of septicaemia and ARDS after HSCT. The present median age of patients survived is 14 years (1-31 years). Genetic analysis was performed in 10 patients. RFX5 homozygous gene defect was found in three patients (P1, P4 and P8), and RFXANK (P2 and P14) and RFXAP (P18 and P19) heterozygous gene defects were found in each two patients, respectively. This large cohort showed that BLS patients have severe combined immunodeficiency (SCID)-like clinical findings. Flow cytometric MHC-II expression study is crucial for the diagnosis, differential diagnosis with SCID, early haematopoietic stem cell transplantation (HSCT), and post-HSCT follow-up. Genetic studies are required first for matched family donor evaluation before HSCT and then for genetic counselling.
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Infecciones por Citomegalovirus , Linfopenia , Inmunodeficiencia Combinada Grave , Humanos , Femenino , Masculino , Adolescente , Turquía , Estudios RetrospectivosRESUMEN
The leishmaniases are NDTs (neglected tropical diseases) that affect people all over the world. They are brought on by protozoans from the genus Leishmania and disseminated by phlebotomine flies that are afflicted with the disease. The best option to manage and lower the incidence of these diseases has been thought by the creation of a safe and effective vaccination. This research used an in silico based mining approach to look for high potential epitopes that might bind to MHC Class I and MHC Class II molecules (mainly; HLA-A*02:01 & HLA-DRB1*03:01) from human population in order to promote vaccine development. Based on the presence of signal peptides, GPI anchors, antigenicity predictions, and a subtractive proteomic technique, we have screened 17 putative antigenic proteins from the 8083 total proteins of L. major. After that thorough immunogenic epitope prediction were done using IEDB-AR tools. We isolated five immunogenic epitopes (three 9-mer & two 15-mer) from five antigenic proteins through docking and MD simulation analysis. Finally, these five anticipated epitopes, viz., TLPEIPVNV, ELMAPVFGL, TLAAAVALL, NSINIRLDGVTSAGF and NVPLVVDASSLFRVA have considerably stronger binding potential with their respective alleles and may trigger immunological responses. The goal of this work was to identify MHC restricted epitopes for CD8+ and CD4+ T cells activation using immunoinformatics in order to identify potential vaccine candidates against L. major parasites.
Asunto(s)
Epítopos de Linfocito T , Leishmania major , Humanos , Epítopos de Linfocito T/química , Leishmania major/metabolismo , Proteoma , Inmunoinformática , Proteómica , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Biología ComputacionalRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Jin-Gui-Shen-Qi Wan (JGSQW) is a traditional Chinese medicine formula that has been traditionally used to alleviate urinary system ailments such as frequent urination and polyuria. Clinical studies have indicated that when combined with hypoglycaemic drugs, JGSQW exhibits a synergistic effect and can improve diabetic nephropathy (DN), yet its underlying mechanism and targets remain unclear. AIM OF THE STUDY: This study aims to investigate the therapeutic efficacy of JGSQW and its underlying mechanisms using a DN db/db mouse model. MATERIALS AND METHODS: Ultrahigh-performance liquid chromatography coupled with mass spectrometry was utilized to analyse the primary active compounds, blood levels, and pharmacokinetics of JGSQW. Additionally, the therapeutic effects of JGSQW and metformin on blood glucose levels, lipid levels, renal function, and renal pathology in diabetic nephropathy mice were investigated using a db/db mouse model. Proteomic analysis was carried out to identify the primary target of JGSQW in treating DN. The mechanism of action was verified by western blotting, immunohistochemistry, and immunofluorescence. Then, molecular docking and molecular dynamics, transfection, drug affinity responsive target stability (DARTS) assay and cell thermal migration assay (CETSA) further validated the targeted binding effect. RESULTS: JGSQW combined with metformin significantly improved the blood glucose levels, blood lipids, renal function, and renal pathology of DN mice. JGSQW mainly exerted its therapeutic effect on DN by targeting major histocompatibility complex class II (MHC class II) molecules. Immunohistochemistry results showed that JGSQW inhibited the expression of collagen I, fibronectin, and alpha smooth muscle actin (α-SMA) expression. Immunofluorescence and Western blot results showed that JGSQW inhibited the expression of H2-Ab1 and H2-Aa, which are MHC class II molecules, thereby suppressing CD4+ T-cell infiltration and improving diabetic kidney fibrosis. The binding ability of paeoniflorin to H2-Aa was predicted and verified by molecular, DARTS, and CETSA assays. Treatment with 80 µM paeoniflorin effectively alleviated high glucose-induced injury in the MPC-5 injury model. H2-Aa was overexpressed at this model concentration, and Western blotting further confirmed that paeoniflorin reduced glomerular podocyte fibrosis by regulating H2-Aa. CONCLUSIONS: JGSQW combined with metformin may have a synergistic effect to alleviates renal fibrosis in diabetic nephropathy by downregulating immune complex MHC class II molecules and attenuating the antigen presentation effect of MHC class II on CD4.