RESUMO
B cell self-tolerance is maintained through multiple checkpoints, including restraints on intracellular signaling and cell trafficking. P2RY8 is a receptor with established roles in germinal center (GC) B cell migration inhibition and growth regulation. Somatic P2RY8 variants are common in GC-derived B cell lymphomas. Here, we identify germline novel or rare P2RY8 missense variants in lupus kindreds or the related antiphospholipid syndrome, including a "de novo" variant in a child with severe nephritis. All variants decreased protein expression, F-actin abundance, and GPCR-RhoA signaling, and those with stronger effects increased AKT and ERK activity and cell migration. Remarkably, P2RY8 was reduced in B cell subsets from some SLE patients lacking P2RY8 gene variants. Low P2RY8 correlated with lupus nephritis and increased age-associated B cells and plasma cells. By contrast, P2RY8 overexpression in cells and mice restrained plasma cell development and reinforced negative selection of DNA-reactive developing B cells. These findings uncover a role of P2RY8 in immunological tolerance and lupus pathogenesis.
Assuntos
Síndrome Antifosfolipídica/imunologia , Tolerância Imunológica/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Mutação de Sentido Incorreto/imunologia , Receptores Purinérgicos P2Y/imunologia , Animais , Síndrome Antifosfolipídica/genética , Síndrome Antifosfolipídica/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Tolerância Imunológica/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/imunologia , Nefrite Lúpica/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto/genética , Linhagem , Plasmócitos/imunologia , Plasmócitos/metabolismo , Receptores Purinérgicos P2Y/genética , Receptores Purinérgicos P2Y/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe.
Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , Mutação de Sentido Incorreto/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Antivirais/sangue , Sítios de Ligação/genética , COVID-19/virologia , Glicosilação , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Manose/metabolismo , Mutação de Sentido Incorreto/genética , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Prolina/genética , Prolina/imunologia , Prolina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Inhibitor of nuclear factor kappa B kinase alpha (IKKα) is critical for p100/NF-κB2 phosphorylation and processing into p52 and activation of the noncanonical NF-κB pathway. A patient with recurrent infections, skeletal abnormalities, absent secondary lymphoid structures, reduced B cell numbers, hypogammaglobulinemia, and lymphocytic infiltration of intestine and liver was found to have a homozygous p.Y580C mutation in the helix-loop-helix domain of IKKα. The mutation preserves IKKα kinase activity but abolishes the interaction of IKKα with its activator NF-κBinducing kinase and impairs lymphotoxin-ßdriven p100/NF-κB2 processing and VCAM1 expression. Homozygous IKKαY580C/Y580C mutant mice phenocopy the patient findings; lack marginal zone B cells, germinal centers, and antigen-specific T cell response to cutaneous immunization; have impaired Il17a expression; and are susceptible to cutaneous Staphylococcus aureus infection. In addition, these mice demonstrate a severe reduction in medullary thymic epithelial cells, impaired thymocyte negative selection, a restricted TCRVß repertoire, a selective expansion of potentially autoreactive T cell clones, a decreased frequency of regulatory T cells, and infiltration of liver, pancreas, and lung by activated T cells coinciding with organ damage. Hence, this study identifies IKKα deficiency as a previously undescribed cause of primary immunodeficiency with associated autoimmunity.
Assuntos
Autoimunidade/imunologia , Quinase I-kappa B/imunologia , Mutação de Sentido Incorreto/genética , Animais , Células HEK293 , Humanos , Quinase I-kappa B/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto/imunologiaRESUMO
Ataxia-telangiectasia (AT) is a rare autosomal recessive neurodegenerative multisystem disorder. A minority of AT patients can present late-onset atypical presentations due to unknown mechanisms. The demographic, clinical, immunological and genetic data were collected by direct interview and examining the Iranian AT patients with late-onset manifestations. We also conducted a systematic literature review for reported atypical AT patients. We identified three Iranian AT patients (3/249, 1.2% of total registry) with later age at ataxia onset and slower neurologic progression despite elevated alpha-fetoprotein levels, history of respiratory infections, and immunological features of the syndrome. Of note, all patients developed autoimmunity in which a decrease of naïve T cells and regulatory T cells were observed. The literature searches also summarized data from 73 variant AT patients with atypical presentation indicating biallelic mild mutations mainly lead to an atypical phenotype with an increased risk of cancer. Variant AT patients present with milder phenotype or atypical form of classical symptoms causing under- or mis- diagnosis. Although missense mutations are more frequent, an atypical presentation can be associated with deleterious mutations due to unknown modifying factors.
Assuntos
Ataxia Telangiectasia/genética , Ataxia/genética , Mutação de Sentido Incorreto/genética , Adolescente , Adulto , Ataxia/imunologia , Ataxia Telangiectasia/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Irã (Geográfico) , Masculino , Mutação de Sentido Incorreto/imunologia , Fenótipo , Linfócitos T Reguladores/imunologia , Adulto Jovem , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/imunologiaRESUMO
Rare autosomal-recessive variants in tetratricopeptide repeat domain 7A (TTC7A) gene have been shown to cause intestinal and immune disorders of variable severity. Missense mutations in TTC7A gene, usually retaining most of the functional motifs, is associated with relative milder clinical presentations. In this study, we reported a patient who was suffering from severe multiple intestinal atresia (MIA) with combined immunodeficiency (CID) that led to the pyloric diaphragm, ileum atresia, colon stenosis, and multiple episodes of sepsis. In spite of several surgeries and supportive treatment, the patient died of severe sepsis and multiple organ failure at age of 3 months. The whole exome sequencing (WES) of peripheral blood samples identified a novel homozygous TTC7A missense mutation (c. 206T>C, p. L69P), inherited from his parents with consanguineous marriage. In silico analysis revealed that a hydrogen bond present between Gly65 and Leu69 in the wild-type TTC7A was disrupted by the Leu69Pro mutation. Moreover, this homozygous missense mutation led to a reduced TTC7A expression in lymphocytes and intestinal tissues, accompanied by impeded lymphocyte development. Further studies demonstrated that the PI4K-FAM126A-EFR3A pathway was impaired in colon tissues. Our data strongly support the linkage of severe MIA-CID with the missense mutation in TTC7A gene. More knowledge of the TTC7A protein functions will have important therapeutic implications for patients with MIA-CID.
Assuntos
Atresia Intestinal/genética , Mutação de Sentido Incorreto/genética , Proteínas/genética , Imunodeficiência Combinada Severa/genética , Criança , Humanos , Atresia Intestinal/imunologia , Masculino , Mutação de Sentido Incorreto/imunologia , Proteínas/imunologia , Imunodeficiência Combinada Severa/imunologiaRESUMO
Guillain-Barré syndrome (GBS), including its variant Miller Fisher syndrome (MFS), is an acute peripheral neuropathy that involves autoimmune mechanisms leading to the production of autoantibodies to gangliosides; sialic acid-containing glycosphingolipids. Although association with various genetic polymorphisms in the major histocompatibility complex (MHC) is shown in other autoimmune diseases, GBS is an exception, showing no such link. No significant association was found by genome wide association studies, suggesting that GBS is not associated with common variants. To address the involvement of rare variants in GBS, we analyzed Siglec-10, a sialic acid-recognizing inhibitory receptor expressed on B cells. Here we demonstrate that two rare variants encoding R47Q and A108V substitutions in the ligand-binding domain are significantly accumulated in patients with GBS. Because of strong linkage disequilibrium, there was no patient carrying only one of them. Recombinant Siglec-10 protein containing R47Q but not A108V shows impaired binding to gangliosides. Homology modeling revealed that the R47Q substitution causes marked alteration in the ligand-binding site. Thus, GBS is associated with a rare variant of the SIGLEC10 gene that impairs ligand binding of Siglec-10. Because Siglec-10 regulates antibody production to sialylated antigens, our finding suggests that Siglec-10 regulates development of GBS by suppressing antibody production to gangliosides, with defects in its function predisposing to disease.
Assuntos
Gangliosídeos/imunologia , Predisposição Genética para Doença , Síndrome de Guillain-Barré/imunologia , Lectinas/imunologia , Mutação de Sentido Incorreto/imunologia , Polimorfismo de Nucleotídeo Único/imunologia , Receptores de Superfície Celular/imunologia , Alelos , Sequência de Aminoácidos , Autoanticorpos/imunologia , Sítios de Ligação/genética , Feminino , Gangliosídeos/metabolismo , Frequência do Gene , Genótipo , Síndrome de Guillain-Barré/genética , Síndrome de Guillain-Barré/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Masculino , Pessoa de Meia-Idade , Síndrome de Miller Fisher/genética , Síndrome de Miller Fisher/imunologia , Síndrome de Miller Fisher/metabolismo , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The R620W polymorphism in protein tyrosine phosphatase nonreceptor type 22 (PTPN22) predisposes carriers to several autoimmune diseases. Two papers in Science Immunology and Science Signaling on this human disease-associated variant lead us to propose a new "switch-of-function" model.
Assuntos
Doenças Autoimunes/imunologia , Modelos Genéticos , Modelos Imunológicos , Mutação de Sentido Incorreto/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/imunologia , Transdução de Sinais/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Predisposição Genética para Doença/genética , Humanos , Camundongos Transgênicos , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , Transdução de Sinais/genéticaRESUMO
CONTEXT: - NRAS is a member of the RAS family oncoproteins implicated in cancer. Gain-of-function NRAS mutations were reported in a subset of colorectal cancers. These mutations occur at codons 12, 13, and 61 and are detected by molecular genetic testing. Recently, an antibody (clone SP174) became available to immunohistochemically pinpoint NRAS Q61R mutant protein. In malignant melanoma, NRAS Q61R mutant-specific immunohistochemistry was shown to be a valuable supplement to traditional genetic testing. OBJECTIVE: - To evaluate the significance of NRAS Q61R mutant-specific immunohistochemistry in a cohort of colorectal carcinomas. DESIGN: - A total of 1185 colorectal carcinomas were immunohistochemically evaluated with SP174 antibody. NRAS Q61R mutant-specific immunohistochemistry was validated by molecular genetic testing including Sanger sequencing, quantitative polymerase chain reaction (qPCR), and next-generation sequencing. RESULTS: - Twelve tumors showed strong SP174 immunoreactivity. Sanger sequencing detected an identical c.182A>G substitution, causing NRAS Q61R mutation at the protein level, only in 8 SP174-positive cases. These results were confirmed by qPCR study. Subsequently, NRAS wild-type tumors with strong SP174 staining were evaluated by next-generation sequencing and revealed KRAS c.182A>G substitutions predicted to cause KRAS Q61R mutation. Review of colorectal carcinomas with known KRAS and NRAS genotype revealed that none of 62 wild-type tumors or 47 mutants other than Q61R were SP174 positive. CONCLUSION: - SP174 immunohistochemistry allows sensitive detection of NRAS and KRAS Q61R mutants. However, molecular genetic testing is necessary to determine specifically which RAS gene is mutated.
Assuntos
Anticorpos/imunologia , Neoplasias Colorretais/imunologia , Reações Cruzadas/imunologia , GTP Fosfo-Hidrolases/imunologia , Proteínas de Membrana/imunologia , Proteínas Mutantes/imunologia , Mutação de Sentido Incorreto/imunologia , Sequência de Bases , Estudos de Coortes , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , GTP Fosfo-Hidrolases/genética , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica/métodos , Proteínas de Membrana/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
Changes associated with the resistance to physical and chemical factors in the hemagglutinin (HA) of influenza A viruses may play an important role in the selection of different influenza variants during circulation in nature. Here, we studied the escape mutants of influenza virus A/mallard/Pennsylvania/10218/84 (H5N2) that were selected by the monoclonal antibody. The escape mutant m4F11(4) carried a single amino acid substitution in large subunit (HA1) of the HA, S145P1, and two ones, m4G10(10) and m4G10(6), had additional amino acid changes in the small subunit (HA2), namely: L124F2 and L124F2 + N79D2, respectively. As it has been found the substitutions appeared in the HA2 of m4G(10) and m4G(6) viruses compensated negative effect of the S145P1 mutation and provided a significant increase in the viral replication ability at the early stage of infection in embryonated chicken eggs as well as in HA thermostability in comparison with m4F11(4) mutant. Phenotypic properties that provide advantages in the process of virus replication can play a role of the positive selection factor in viral population.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A Subtipo H5N2 , Mutação de Sentido Incorreto/imunologia , Substituição de Aminoácidos , Animais , Embrião de Galinha , Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H5N2/genética , Vírus da Influenza A Subtipo H5N2/imunologia , Influenza Aviária/genética , Influenza Aviária/imunologiaRESUMO
Human immunoglobulin G isotype 4 (IgG4) antibodies (Abs) are potential candidates for immunotherapy when reduced effector functions are desirable. IgG4 Abs are dynamic molecules able to undergo a process known as Fab arm exchange (FAE). This results in functionally monovalent, bispecific antibodies (bsAbs) with unknown specificity and hence, potentially, reduced therapeutic efficacy. IgG4 FAE is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 Abs. To date, the mechanism of FAE is not entirely understood and studies measuring FAE in ex vivo matrices have been hampered by the presence and abundance of endogenous IgG4 wild-type (WT) Abs. Using representative humanized WT IgG4 monoclonal Abs, namely, anti-IL-6 and anti-TNF, and a core-hinge stabilized serine 228 to proline (S228P) anti-IL-6 IgG4 mutant, it is demonstrated for the first time how anti-IgG4 affinity chromatography can be used to prepare physiologically relevant matrices for assessing and quantifying FAE. A novel method for quantifying FAE using a single MSD immunoassay is also reported and confirms previous findings that, dependent on the redox conditions, the S228P mutation can prevent IgG4 FAE to undetectable levels both in vitro and in vivo. Together, the findings and novel methodologies will allow researchers to monitor and quantify FAE of their own IgG4 molecules in physiologically relevant matrices.
Assuntos
Fragmentos Fab das Imunoglobulinas/genética , Imunoglobulina G/genética , Mutação de Sentido Incorreto/genética , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Western Blotting , Humanos , Fragmentos Fab das Imunoglobulinas/sangue , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Mutação de Sentido Incorreto/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Cancer genome sequencing has enabled the rapid identification of the complete repertoire of coding sequence mutations within a patient's tumor and facilitated their use as personalized immunogens. Although a variety of techniques are available to assist in the selection of mutation-defined epitopes to be included within the tumor vaccine, the ability of the peptide to bind to patient MHC is a key gateway to peptide presentation. With advances in the accuracy of predictive algorithms for MHC class I binding, choosing epitopes on the basis of predicted affinity provides a rapid and unbiased approach to epitope prioritization. We show herein the retrospective application of a prediction algorithm to a large set of bona fide T cell-defined mutated human tumor antigens that induced immune responses, most of which were associated with tumor regression or long-term disease stability. The results support the application of this approach for epitope selection and reveal informative features of these naturally occurring epitopes to aid in epitope prioritization for use in tumor vaccines.
Assuntos
Antígenos de Neoplasias/genética , Epitopos de Linfócito T/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias/imunologia , Algoritmos , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/genética , Humanos , Imunidade Celular/genética , Imunidade Celular/imunologia , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/imunologia , Neoplasias/genética , Linfócitos T/imunologiaRESUMO
INTRODUCTION: The majority of the genetic variance of systemic lupus erythematosus (SLE) remains unexplained by the common disease-common variant hypothesis. Rare variants, which are not detectable by genome-wide association studies because of their low frequencies, are predicted to explain part of this "missing heritability." However, recent studies identifying rare variants within known disease-susceptibility loci have failed to show genetic associations because of their extremely low frequencies, leading to the questioning of the contribution of rare variants to disease susceptibility. A common (minor allele frequency = 17.4% in cases) nonsynonymous coding variant rs1143679 (R77H) in ITGAM (CD11b), which forms half of the heterodimeric integrin receptor, complement receptor 3 (CR3), is robustly associated with SLE and has been shown to impair CR3-mediated phagocytosis. METHODS: We resequenced ITGAM in 73 SLE cases and identified two previously unidentified, case-specific nonsynonymous variants, F941V and G1145S. Both variants were genotyped in 2,107 and 949 additional SLE cases, respectively, to estimate their frequencies in a disease population. An in vitro model was used to assess the impact of F941V and G1145S, together with two nonsynonymous ITGAM polymorphisms, A858V (rs1143683) and M441T (rs11861251), on CR3-mediated phagocytosis. A paired two-tailed t test was used to compare the phagocytic capabilities of each variant with that of wild-type CR3. RESULTS: Both rare variants, F941V and G1145S, significantly impair CR3-mediated phagocytosis in an in vitro model (61% reduction, P = 0.006; 26% reduction, P = 0.0232). However, neither of the common variants, M441T and A858V, had an effect on phagocytosis. Neither rare variant was observed again in the genotyping of additional SLE cases, suggesting that their frequencies are extremely low. CONCLUSIONS: Our results add further evidence to the functional importance of ITGAM in SLE pathogenesis through impaired phagocytosis. Additionally, this study provides a new example of the identification of rare variants in common-allele-associated loci, which, because of their extremely low frequencies, are not statistically associated. However, the demonstration of their functional effects adds support to their contribution to disease risk, and questions the current notion of dismissing the contribution of very rare variants on purely statistical analyses.
Assuntos
Antígeno CD11b/genética , Predisposição Genética para Doença/genética , Lúpus Eritematoso Sistêmico/genética , Mutação de Sentido Incorreto/genética , Animais , Sequência de Bases , Antígeno CD11b/imunologia , Células COS , Chlorocebus aethiops , Citometria de Fluxo , Frequência do Gene , Genótipo , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/imunologia , Mutação de Sentido Incorreto/imunologia , Fagocitose/genética , Fagocitose/imunologia , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
Surveillance data indicate that most circulating A(H1N1)pdm09 influenza viruses have remained antigenically similar since they emerged in humans in 2009. However, antigenic drift is likely to occur in the future in response to increasing population immunity induced by infection or vaccination. In this study, sequential passaging of A(H1N1)pdm09 virus by contact transmission through two independent series of suboptimally vaccinated ferrets resulted in selection of variant viruses with an amino acid substitution (N156K, H1 numbering without signal peptide; N159K, H3 numbering without signal peptide; N173K, H1 numbering from first methionine) in a known antigenic site of the viral HA. The N156K HA variant replicated and transmitted efficiently between naïve ferrets and outgrew wildtype virus in vivo in ferrets in the presence and absence of immune pressure. In vitro, in a range of cell culture systems, the N156K variant rapidly adapted, acquiring additional mutations in the viral HA that also potentially affected antigenic properties. The N156K escape mutant was antigenically distinct from wildtype virus as shown by binding of HA-specific antibodies. Glycan binding assays demonstrated the N156K escape mutant had altered receptor binding preferences compared to wildtype virus, which was supported by computational modeling predictions. The N156K substitution, and culture adaptations, have been detected in human A(H1N1)pdm09 viruses with N156K preferentially reported in sequences from original clinical samples rather than cultured isolates. This study demonstrates the ability of the A(H1N1)pdm09 virus to undergo rapid antigenic change to evade a low level vaccine response, while remaining fit in a ferret transmission model of immunization and infection. Furthermore, the potential changes in receptor binding properties that accompany antigenic changes highlight the importance of routine characterization of clinical samples in human A(H1N1)pdm09 influenza surveillance.
Assuntos
Antígenos Virais , Deriva Genética , Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Mutação de Sentido Incorreto/imunologia , Pandemias , Substituição de Aminoácidos , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Embrião de Galinha , Modelos Animais de Doenças , Cães , Feminino , Furões , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/epidemiologia , Influenza Humana/genética , Influenza Humana/imunologia , Células Madin Darby de Rim Canino , MasculinoRESUMO
PURPOSE: Immunological and molecular evaluation of a patient presenting with recurrent infections caused by Streptococcus pneumoniae and low complement component 3 (C3) levels. METHODS: Immunological evaluation included complement components and immunoglobulin level quantification as well as number and function of T cells, B cells and neutrophils. Serotype-specific immunoglobulin G antibodies against S. pneumoniae capsular polysaccharides were quantified by ELISA in serum samples before and after vaccination with unconjugated polysaccharide vaccine. For the molecular analysis, genomic DNA from the patient and parents were isolated and all exons as well as exon-intron boundaries of the C3 gene were sequenced by Sanger sequencing. RESULTS: A 16-year-old male, born to consanguineous parents, presented with recurrent episodes of pneumonia caused by S. pneumoniae and bronchiectasis. The patient showed severely reduced C3 and immunoglobulin A levels, while the parents showed moderately reduced levels of C3. Mutational analysis revealed a novel, homozygous missense mutation in the C3 gene (c. C4554G, p. Cys1518Trp), substituting a highly conserved amino acid in the C345C domain of C3 and interrupting one of its disulfide bonds. Both parents were found to be carriers of the affected allele. Vaccination against S. pneumoniae resulted in considerable clinical improvement. CONCLUSIONS: We report a novel homozygous mutation in the C3 gene in a patient with concomitant selective IgA deficiency who presented with a marked clinical improvement after vaccination against S. pneumoniae. This observation underlines the notion that vaccination against this microorganism is an important strategy for treatment of PID patients, particularly those presenting with increased susceptibility to infections caused by this agent.
Assuntos
Complemento C3/genética , Deficiência de IgA/genética , Deficiência de IgA/imunologia , Mutação de Sentido Incorreto , Adolescente , Bronquiectasia/complicações , Bronquiectasia/genética , Bronquiectasia/imunologia , Criança , Pré-Escolar , Comorbidade , Complemento C3/antagonistas & inibidores , Complemento C3/biossíntese , Feminino , Humanos , Deficiência de IgA/complicações , Masculino , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/imunologia , Linhagem , Vacinas Pneumocócicas/uso terapêutico , Pneumonia Pneumocócica/genética , Pneumonia Pneumocócica/imunologia , Pneumonia Pneumocócica/prevenção & controle , Mutação Puntual/genética , Mutação Puntual/imunologia , Homologia de Sequência de AminoácidosRESUMO
PURPOSE: Acute Hemorrhagic Leukoencephalitis (AHLE) is a rare demyelinating disorder of acute onset, rapid deterioration and significant morbidity and mortality. Most often described as a post-infectious complication of an upper respiratory illness, its precise pathophysiology remains unclear. We describe two pediatric patients with AHLE with partial complement factor I (FI) deficiency whose successful treatment included the interleukin-1 (IL-1) receptor antagonist, anakinra, implicating a role for FI and IL-1 in this disorder. METHODS: Extensive clinical workup of two patients presenting with AHLE revealed complement abnormalities, specifically related to the alternative pathway and its regulator, FI. Aggressive management with steroids, immunoglobulin, and anakinra ultimately led to improvement of clinical status and near return to neurologic baseline in both patients. Genetic sequencing of the FI coding regions of the patients and their families was performed. In vitro protein expression studies and immunohistochemistry of fixed brain tissue was used to investigate pathogenic mechanisms. RESULTS: Two novel mutations in FI were identified in our patients, which result in failure to secrete FI. Immunohistochemical evaluation of brain tissue demonstrated positive staining for C3, membrane attack complex (MAC) and IL-1. CONCLUSIONS: We propose AHLE is an unreported, rare phenotype for partial FI deficiency. The upregulation of C3, MAC and IL-1 with subsequent demyelination support a pathologic role for complement activation in AHLE, and suggest anakinra as an important adjunctive therapy in this disease.
Assuntos
Fator I do Complemento/genética , Leucoencefalite Hemorrágica Aguda/genética , Leucoencefalite Hemorrágica Aguda/imunologia , Mutação de Sentido Incorreto/imunologia , Neurônios/imunologia , Neurônios/patologia , Adolescente , Adulto , Criança , Ativação do Complemento/genética , Ativação do Complemento/imunologia , Complemento C3/fisiologia , Fator I do Complemento/deficiência , Fator I do Complemento/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/fisiologia , Feminino , Células HEK293 , Humanos , Imunofenotipagem , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-1/fisiologia , Leucoencefalite Hemorrágica Aguda/patologia , Masculino , Neurônios/metabolismo , LinhagemRESUMO
The microtubule-associated protein Tau plays a critical role in the pathogenesis of Alzheimer disease and several related disorders (tauopathies). In the disease Tau aggregates and becomes hyperphosphorylated forming paired helical and straight filaments, which can further condense into higher order neurofibrillary tangles in neurons. The development of this pathology is consistently associated with progressive neuronal loss and cognitive decline. The identification of tractable therapeutic targets in this pathway has been challenging, and consequently very few clinical studies addressing Tau pathology are underway. Recent active immunization studies have raised the possibility of modulating Tau pathology by activating the immune system. Here we report for the first time on passive immunotherapy for Tau in two well established transgenic models of Tau pathogenesis. We show that peripheral administration of two antibodies against pathological Tau forms significantly reduces biochemical Tau pathology in the JNPL3 mouse model. We further demonstrate that peripheral administration of the same antibodies in the more rapidly progressive P301S tauopathy model not only reduces Tau pathology quantitated by biochemical assays and immunohistochemistry, but also significantly delays the onset of motor function decline and weight loss. This is accompanied by a reduction in neurospheroids, providing direct evidence of reduced neurodegeneration. Thus, passive immunotherapy is effective at preventing the buildup of intracellular Tau pathology, neurospheroids, and associated symptoms, although the exact mechanism remains uncertain. Tau immunotherapy should therefore be considered as a therapeutic approach for the treatment of Alzheimer disease and other tauopathies.
Assuntos
Doença de Alzheimer/terapia , Anticorpos/imunologia , Anticorpos/farmacologia , Imunização Passiva/métodos , Proteínas tau/imunologia , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Substituição de Aminoácidos/imunologia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Atividade Motora/imunologia , Mutação de Sentido Incorreto/imunologia , Proteínas tau/genéticaRESUMO
Hereditary complete deficiency of complement component C1q is associated with a high prevalence of systemic lupus erythematosus and increased susceptibility to severe recurrent infections. An 11-year-old girl was screened for immunodeficiency due to a history of recurrent meningitis and pneumonia. Immunologic studies revealed absence of classic pathway hemolytic activity and undetectable levels of Clq. Exon-specific amplification of genomic DNA by polymerase chain reaction followed by direct sequence analysis revealed a novel homozygous missense mutation at codon 48 in the C1q C gene causing a glycine-to-arginine substitution affecting the collagen-like region of C1q. No changes were seen in the exons of the A and B chains. The mutation affected both the formation and the secretion of C1q variant molecules. We describe a novel mutation in the C1q C chain gene that leads to an interchange in amino acids resulting in absence of C1q in serum.
Assuntos
Complemento C1q/deficiência , Complemento C1q/genética , Criança , Complemento C1q/imunologia , Via Clássica do Complemento/genética , Via Clássica do Complemento/imunologia , DNA/química , DNA/genética , Feminino , Homozigoto , Humanos , Masculino , Mutação de Sentido Incorreto/imunologia , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , TurquiaRESUMO
BACKGROUND: In the acute phase of infection with feline immunodeficiency virus (FIV), the virus targets activated CD4+ T cells by utilising CD134 (OX40) as a primary attachment receptor and CXCR4 as a co-receptor. The nature of the virus-receptor interaction varies between isolates; strains such as GL8 and CPGammer recognise a "complex" determinant on CD134 formed by cysteine-rich domains (CRDs) 1 and 2 of the molecule while strains such as PPR and B2542 require a more "simple" determinant comprising CRD1 only for infection. These differences in receptor recognition manifest as variations in sensitivity to receptor antagonists. In this study, we ask whether the nature of the virus-receptor interaction evolves in vivo. RESULTS: Following infection with a homogeneous viral population derived from a pathogenic molecular clone, a quasispecies emerged comprising variants with distinct sensitivities to neutralising antibody and displaying evidence of conversion from a "complex" to a "simple" interaction with CD134. Escape from neutralising antibody was mediated primarily by length and sequence polymorphisms in the V5 region of Env, and these alterations in V5 modulated the virus-receptor interaction as indicated by altered sensitivities to antagonism by both anti-CD134 antibody and soluble CD134. CONCLUSIONS: The FIV-receptor interaction evolves under the selective pressure of the host humoral immune response, and the V5 loop contributes to the virus-receptor interaction. Our data are consistent with a model whereby viruses with distinct biological properties are present in early versus late infection and with a shift from a "complex" to a "simple" interaction with CD134 with time post-infection.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Síndrome de Imunodeficiência Adquirida Felina/virologia , Vírus da Imunodeficiência Felina/imunologia , Mutação de Sentido Incorreto/imunologia , Proteínas do Envelope Viral/imunologia , Ligação Viral , Animais , Gatos , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Felina/isolamento & purificação , Vírus da Imunodeficiência Felina/fisiologia , Modelos Moleculares , Conformação Proteica , Receptores Virais/metabolismo , Seleção Genética , Proteínas do Envelope Viral/genéticaRESUMO
Two missense variants (D299G and T399I) of TLR4 are cosegregated in individuals of European descent and, in a number of test systems, result in reduced responsiveness to endotoxin. How these changes within the ectodomain (ecd) of TLR4 affect TLR4 function is unclear. For both wild-type and D299G.T399I TLR4, we used endotoxinCD14 and endotoxinMD-2 complexes of high specific radioactivity to measure: 1) interaction of recombinant MD-2TLR4 with endotoxinCD14 and TLR4 with endotoxinMD-2; 2) expression of functional MD-2TLR4 and TLR4; and 3) MD-2TLR4 and TLR4-dependent cellular endotoxin responsiveness. Both wild-type and D299G.T399I TLR4(ecd) demonstrated high affinity (K(d) approximately 200 pM) interaction of endotoxinCD14 with MD-2TLR4(ecd) and endotoxinMD-2 with TLR4(ecd). However, levels of functional TLR4 were reduced up to 2-fold when D299G.T399I TLR4 was coexpressed with MD-2 and >10-fold when expressed without MD-2, paralleling differences in cellular endotoxin responsiveness. The dramatic effect of the D299G.T399I haplotype on expression of functional TLR4 without MD-2 suggests that cells expressing TLR4 without MD-2 are most affected by these polymorphisms.