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1.
BMC Bioinformatics ; 21(1): 422, 2020 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-32993478

RESUMO

BACKGROUND: Antigen receptors are characterized by an extreme diversity of specificities, which poses major computational and analytical challenges, particularly in the era of high-throughput immunoprofiling by next generation sequencing (NGS). The T cell Receptor/Immunoglobulin Profiler (TRIP) tool offers the opportunity for an in-depth analysis based on the processing of the output files of the IMGT/HighV-Quest tool, a standard in NGS immunoprofiling, through a number of interoperable modules. These provide detailed information about antigen receptor gene rearrangements, including variable (V), diversity (D) and joining (J) gene usage, CDR3 amino acid and nucleotide composition and clonality of both T cell receptors (TR) and B cell receptor immunoglobulins (BcR IG), and characteristics of the somatic hypermutation within the BcR IG genes. TRIP is a web application implemented in R shiny. RESULTS: Two sets of experiments have been performed in order to evaluate the efficiency and performance of the TRIP tool. The first used a number of synthetic datasets, ranging from 250k to 1M sequences, and established the linear response time of the tool (about 6 h for 1M sequences processed through the entire BcR IG data pipeline). The reproducibility of the tool was tested comparing the results produced by the main TRIP workflow with the results from a previous pipeline used on the Galaxy platform. As expected, no significant differences were noted between the two tools; although the preselection process seems to be stricter within the TRIP pipeline, about 0.1% more rearrangements were filtered out, with no impact on the final results. CONCLUSIONS: TRIP is a software framework that provides analytical services on antigen receptor gene sequence data. It is accurate and contains functions for data wrangling, cleaning, analysis and visualization, enabling the user to build a pipeline tailored to their needs. TRIP is publicly available at https://bio.tools/TRIP_-_T-cell_Receptor_Immunoglobulin_Profiler .


Assuntos
Imunoglobulinas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Interface Usuário-Computador , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulinas/química , Imunoglobulinas/genética , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética
2.
PLoS One ; 15(8): e0236704, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790777

RESUMO

The hepatitis B virus (HBV) envelope is composed of a lipid bilayer and three glycoproteins, referred to as the large (L), middle (M), and small (S) hepatitis B virus surface antigens (HBsAg). S protein constitutes the major portion of the viral envelope and an even greater proportion of subviral particles (SVP) that circulate in the blood. Recombinant S proteins are currently used as a preventive vaccine, while plasma fractions isolated from vaccinated people, referred to as hepatitis B immune globulin (HBIG), are used for short-term prophylaxis. Here, we characterized a recombinant human IgG1 type anti-S antibody named Lenvervimab regarding its binding property to a variety of cloned S antigens. Immunochemical data showed an overall consistent avidity of the antibody to S antigens of most viral genotypes distributed worldwide. Further, antibody binding was not affected by the mutations in the antigenic 'a' determinant found in many clinical variants, including the immune escape mutant G145R. In addition, mutations in the S gene sequence that confer drug resistance to the viral polymerase did not interfere with the antibody binding. These results support for a preventive use of the antibody against HBV infection.


Assuntos
Anticorpos Anti-Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Imunoglobulinas/imunologia , Sequência de Aminoácidos , Reações Antígeno-Anticorpo , Linhagem Celular , Farmacorresistência Viral , Genótipo , Células Hep G2 , Hepatite B/patologia , Hepatite B/virologia , Anticorpos Anti-Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
3.
Proc Natl Acad Sci U S A ; 117(33): 20100-20108, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32727902

RESUMO

Mutation of HELLS (Helicase, Lymphoid-Specific)/Lsh in human DNA causes a severe immunodeficiency syndrome, but the nature of the defect remains unknown. We assessed here the role of Lsh in hematopoiesis using conditional Lsh knockout mice with expression of Mx1 or Vav Cre-recombinase. Bone marrow transplantation studies revealed that Lsh depletion in hematopoietic stem cells severely reduced B cell numbers and impaired B cell development in a hematopoietic cell-autonomous manner. Lsh-deficient mice without bone marrow transplantation exhibited lower Ig levels in vivo compared to controls despite normal peripheral B cell numbers. Purified B lymphocytes proliferated normally but produced less immunoglobulins in response to in vitro stimulation, indicating a reduced capacity to undergo class switch recombination (CSR). Analysis of germline transcripts, examination of double-stranded breaks using biotin-labeling DNA break assay, and End-seq analysis indicated that the initiation of the recombination process was unscathed. In contrast, digestion-circularization PCR analysis and high-throughput sequencing analyses of CSR junctions and a chromosomal break repair assay indicated an impaired ability of the canonical end-joining pathway in Lsh-deficient B cells. Our data suggest a hematopoietic cell-intrinsic role of Lsh in B cell development and in CSR providing a potential target for immunodeficiency therapy.


Assuntos
Linfócitos B/fisiologia , DNA Helicases/metabolismo , Imunoglobulinas/metabolismo , Animais , Linhagem Celular , DNA Helicases/genética , Inativação Gênica , Humanos , Imunoglobulinas/genética , Camundongos , Camundongos Knockout , Mutação
4.
Sci Rep ; 10(1): 9426, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32523038

RESUMO

Keratoconus (KC) is classically considered a non-inflammatory condition caused by central corneal thinning that leads to astigmatism and reduced visual acuity. Previous studies have identified increased systemic levels of pro-inflammatory factors, including interleukin-6, tumor necrosis factor-α, and matrix metalloproteinase-9, suggesting that KC may have an inflammatory component in at least a subset of patients. In this study, we evaluated the levels of different immunoglobulins (light and heavy chains) based on Ig α, Ig λ, Ig κ, Ig µ, and Ig heavy chain subunits in non-KC tears (n = 7 control individuals) and KC tears (n = 7 KC patients) using tandem-liquid chromatography mass spectrometry. The most abundant Ig heavy chains detected in both control individuals and KC patients were Ig α-1 and Ig α-2 likely correlating to the higher IgA levels reported in human tears. We identified significant differences in immunoglobulin κ-chain V-II levels in KC patients compared to control individuals with no significant difference in Ig κ/Ig λ ratios or heavy chain levels. Our study supports previous findings suggesting that KC possesses a systemic component that may contribute to the KC pathology. Further studies are required to define causality and establish a role for systemic immune system-dependent factors and pro-inflammatory processes in KC development or progression.


Assuntos
Proteínas do Olho/metabolismo , Imunoglobulinas/metabolismo , Ceratocone/metabolismo , Lágrimas/metabolismo , Cromatografia Líquida/métodos , Estudos de Coortes , Progressão da Doença , Humanos
5.
Transfusion ; 60(5): 1060-1068, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32369193

RESUMO

BACKGROUND: Anti-red blood cell (RBC) alloantibodies consisting of only the immunoglobulin G (IgG) 4 subtype are typically considered clinically insignificant. A US Food and Drug Administration-approved monoclonal anti-human globulin (16H8) is nonreactive with IgG4, which has been considered a benefit to avoid testing interference from IgG4. However, 16H8 also does not recognize two natural IgG3 variants (IgG3-03 and IgG3-13). Thus, 16H8 may miss clinically significant alloantibodies in some settings. STUDY DESIGN AND METHODS: Novel mouse anti-human IgG hybridomas were generated and screened for reactivity with 32 human variants of anti-KEL1 across different IgG subtypes, as well as mutants to allow epitope mapping. Anti-IgG reactivity was determined using KEL1+ RBCs bound by each IgG variant as targets. Binding of anti-IgG was determined by flow cytometry. RESULTS: 16H8 recognized an epitope involving amino acid 419, which is glutamate in IgG4, IgG3-03, and IgG3-13, explaining the lack of 16H8 reactivity with these subtypes/isoallotypes. A new monoclonal antibody (PUMA8) was isolated that, like 16H8, was nonreactive with IgG4 but without blind spots for known variants of IgG1, IgG2, or IgG3. PUMA8 recognized an epitope containing arginine at position 355, which is glutamine in IgG4. However, a recently described new IgG4 variant with an arginine at position 355 results in PUMA8 reactivity. CONCLUSION: PUMA8 represents an alternative to 16H8 that avoids IgG4 but without blind spots for IgG3 variants. However, PUMA8 reacts with one recently described IgG4 variant. In addition to relevance to immunohematology, these studies highlight the importance of patient variation with regards to assay performance in an era of personalized medicine.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Imunoglobulinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Eritrócitos/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Testes Imunológicos , Isoanticorpos/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Análise de Sequência de Proteína
7.
Nat Commun ; 11(1): 2032, 2020 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-32341344

RESUMO

TENT5C is a non-canonical cytoplasmic poly(A) polymerase highly expressed by activated B cells to suppress their proliferation. Here we measure the global distribution of poly(A) tail lengths in responsive B cells using a Nanopore direct RNA-sequencing approach, showing that TENT5C polyadenylates immunoglobulin mRNAs regulating their half-life and consequently steady-state levels. TENT5C is upregulated in differentiating plasma cells by innate signaling. Compared with wild-type, Tent5c-/- mice produce fewer antibodies and have diminished T-cell-independent immune response despite having more CD138high plasma cells as a consequence of accelerated differentiation. B cells from Tent5c-/- mice also have impaired capacity of the secretory pathway, with reduced ER volume and unfolded protein response. Importantly, these functions of TENT5C are dependent on its enzymatic activity as catalytic mutation knock-in mice display the same defect as Tent5c-/-. These findings define the role of the TENT5C enzyme in the humoral immune response.


Assuntos
Imunidade Humoral , Imunoglobulinas/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Linfócitos B/enzimologia , Diferenciação Celular , Feminino , Regulação Enzimológica da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Nucleotidiltransferases/genética , Fenótipo , RNA-Seq , Transdução de Sinais , Resposta a Proteínas não Dobradas
8.
J Med Life ; 13(1): 21-25, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32341696

RESUMO

Immunopathogenesis of inflammatory and dystrophic diseases of the tissues of the oral cavity is characterized by cellular and humoral factors of specific and nonspecific resistance, the functioning of which is determined by the overall somatic state. This study aimed to study the features of protective mechanisms of the oral cavity due to orthodontic pathology, pathology of periodontal tissues, and odontogenic inflammatory process in children with diffuse nontoxic goiter. Eighty children with diffuse nontoxic goiter aged 12-15 years with different dental status were examined. Evaluation of local immunity of the oral cavity was carried out by determining the content of sIgA, IgA, IgG, lysozyme activity, and levels of IL-1ß, IL-4 by enzyme immunoassay. Immunological studies have shown that in children with diffuse nontoxic goiter, the activity of lysozyme in the oral fluid is decreased. The level of sIgА is also reduced by about 20%. Besides, there is an increase in the content of IgG and a growing trend in the level of IgА. The content of IL-1ß and IL-4 in such children fluctuates more compared to somatically healthy children. In conclusion, a violation of the local protective mechanisms of the oral cavity is observed in children with diffuse nontoxic goiter. Also, the increase in the severity of dental pathology leads to increased tension of local protective and compensatory reactions.


Assuntos
Bócio/patologia , Boca/patologia , Adolescente , Líquidos Corporais/metabolismo , Criança , Humanos , Imunoglobulinas/metabolismo , Interleucina-1beta/metabolismo , Interleucina-4/metabolismo , Masculino
9.
Poult Sci ; 99(4): 1914-1920, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32241471

RESUMO

In avian species, maternal immunoglobulin Y (IgY) is transferred from the blood to the yolks of maturing oocytes; however, the mechanism underlying this transfer is unknown. To gain insight into the mechanisms of maternal IgY transfer into egg yolks, IgY-depleted chickens were generated by removing the bursa of Fabricius (bursectomy) during egg incubation, and their egg production and IgY transport ability into egg yolks were determined. After hatching, blood IgY concentrations of the bursectomized chickens decreased gradually until sexual maturity, whereas those of IgA remained low from an early stage of growth (from at least 2 wk of age). Chickens identified as depleted in IgY through screening of blood IgY and IgA concentrations were raised to sexual maturity. At 20 wk of age, both blood and egg yolk IgY concentrations in the IgY-depleted group were 600-fold lower than those of the control group, whereas egg production did not differ between the groups. Intravenously injected, digoxigenin-labeled IgY uptake into the egg yolk was approximately 2-fold higher in the IgY-depleted chickens than in the controls, suggesting that IgY depletion may enhance IgY uptake in maturing oocytes. DNA microarray analysis of the germinal disc, including the oocyte nucleus, revealed that the expression levels of 73 genes were upregulated more than 1.5-fold in the IgY-depleted group, although we could not identify a convincing candidate gene for the IgY receptor. In conclusion, we successfully raised IgY-depleted chickens presenting a marked reduction in egg yolk IgY. The enhanced uptake of injected IgY into the egg yolks of the IgY-depleted chickens supports the existence of a selective IgY transport mechanism in maturing oocytes and ovarian follicles in avian species.


Assuntos
Proteínas Aviárias/metabolismo , Galinhas/metabolismo , Gema de Ovo/metabolismo , Imunoglobulinas/metabolismo , Animais , Proteínas Aviárias/deficiência , Bolsa de Fabricius/cirurgia , Galinhas/cirurgia , Feminino , Imunoglobulinas/deficiência
10.
Proc Natl Acad Sci U S A ; 117(17): 9393-9400, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32295885

RESUMO

Sperm-oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm-oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm-oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm-oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm-oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm-oocyte fusion process.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Sequência de Aminoácidos , Animais , Fertilização In Vitro , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Oócitos/fisiologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Interações Espermatozoide-Óvulo/fisiologia
11.
Int J Mol Sci ; 21(5)2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32121610

RESUMO

Several studies have suggested that there is a link between membrane attack complex (MAC) deposition in the retina and the progression of diabetic retinopathy (DR). Our recent investigation demonstrated that circulating IgG-laden extracellular vesicles contribute to an increase in retinal vascular permeability in DR through activation of the complement system. However, the mechanism through which extracellular vesicle-induced complement activation contributes to retinal vascular cytolytic damage in DR is not well understood. In this study, we demonstrate that IgG-laden extracellular vesicles in rat plasma activate the classical complement pathway, and in vitro Streptozotocin (STZ)-induced rat diabetic plasma results in MAC deposition and cytolytic damage in human retinal endothelial cells (HRECs). Moreover, removal of the plasma extracellular vesicles reduced the MAC deposition and abrogated cytolytic damage seen in HRECs. Together, the results of this study demonstrate that complement activation by IgG-laden extracellular vesicles in plasma could lead to MAC deposition and contribute to endothelium damage and progression of DR.


Assuntos
Ativação do Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Células Endoteliais/patologia , Vesículas Extracelulares/metabolismo , Retina/patologia , Animais , Morte Celular , Sobrevivência Celular , Complemento C1/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Vesículas Extracelulares/ultraestrutura , Humanos , Imunoglobulinas/metabolismo , Masculino , Ratos Sprague-Dawley
12.
Clin Chem ; 66(3): 445-454, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32031592

RESUMO

BACKGROUND: Despite well-described analytical effects of autoantibodies against cardiac troponin (cTn) I on experimental assays, no study has systematically examined their impact on cTn assays in clinical use. We determined the effects of endogenous antibodies on 5 different cTnI assays and a cTnT assay. METHODS: cTn was measured by 6 methods: Siemens hs-cTnI Centaur, Siemens hs-cTnI Vista, Abbott hs-cTnI Architect, Beckman hs-cTnI Access, Beckman cTnI Access, and Roche hs-cTnT Elecsys. Measurements were repeated on 5 assays (all except Siemens hs-cTnI Vista) following immunoglobulin depletion by incubation with protein A. Low recovery of cTnI (<40%) following immunoglobulin depletion was considered positive for macro-cTnI. Protein A findings were validated by gel filtration chromatography and polyethylene glycol precipitation. RESULTS: In a sample of 223 specimens selected from a community laboratory that uses the Siemens hs-cTnI Centaur assay and from which cTn was requested, 76% of samples demonstrated increased cTnI (median, 88 ng/L; interquartile range, 62-204 ng/L). Macro-cTnI was observed in 123 (55%) of the 223 specimens. Comparisons of cTnI assays markedly improved once patients with macro-cTnI were removed. Passing-Bablok regression analysis between hs-cTnI assays demonstrated different slopes for patients with and without macro-cTnI. In patients with macro-cTnI, 89 (72%) showed no effect on the recovery of cTnT, whereas 34 (28%) had reduced recovery of cTnT. The proportion of results above the manufacturers' 99th percentile varied with the cTn assay and macro-cTnI status. CONCLUSION: We suggest that the observed discrepancy between hs-cTnI assays may be attributed in part to the presence of macro-cTnI.


Assuntos
Bioensaio/métodos , Troponina I/sangue , Troponina T/sangue , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Precipitação Química , Cromatografia em Gel , Humanos , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Kit de Reagentes para Diagnóstico , Análise de Regressão , Proteína Estafilocócica A/metabolismo , Troponina I/isolamento & purificação , Troponina T/isolamento & purificação
13.
Artigo em Inglês | MEDLINE | ID: mdl-32062365

RESUMO

Antigen-binding (Fab) and crystallizable (Fc) fragments are the active components of yolk immunoglobulin (IgY), which have been widely used in the pharmaceutical field. However, the common purification methods for the Fab and Fc fragments use combinations of multi-columns are complex and time-consuming. The objective of this study was to improve the separation efficiency of the Fab and Fc fragments from the hydrolyzed IgY and increase the purity of the isolated Fab and Fc fragments. Natural IgY was hydrolyzed using papain for 6 hr and then treated with 45% saturated ammonium sulfate to remove small molecular-weight-peptides. The fraction containing Fab and Fc fragments was loaded on a DEAE-Sepharose ion exchange column and the Fab fraction was washed out first with 10 mM Tris-HCl buffer (pH 7.6). Then, the Fc fraction bound to the DEAE Sepharose was eluted with 10 mM Tris-HCl buffer (pH 7.6) containing 0.21 M NaCl. The purity of the two fragments was 88.7% and 90.1%, respectively. The results of Western blotting and MS analyses indicated that this method purified Fab and Fc fractions with high purity. This method is easy and simple compared with other methods, and the active fragments separated can be easily used.


Assuntos
Fragmentos Fab das Imunoglobulinas/análise , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/análise , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulinas/metabolismo , Sulfato de Amônio/química , Animais , Western Blotting , Galinhas , Cromatografia por Troca Iônica , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulinas/química , Papaína/metabolismo
14.
Sci Rep ; 10(1): 342, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941968

RESUMO

In carcinomas, the nature of CD40 ligand shapes the outcome of CD40 ligation. To date, the consequences of membrane-bound CD40L (mCD40L) on its immune-stimulatory function are unknown. Here, we examined the impact of mCD40L versus soluble CD40L (sCD40L) on T24 bladder carcinoma gene expression profiling. Of 410 differentially expressed genes, 286 were upregulated and 124 downregulated by mCD40L versus sCD40L. Gene ontology enrichment analysis revealed immune-stimulatory function as the most significant enriched biological process affected by upregulated transcripts, while those downregulated were critical for cell growth and division. Furthermore, immature dendritic cells (iDC) responded to mCD40L with enhanced maturation and activation over sCD40L evidenced by higher expression levels of CD83, CD86, HLA-DR and CD54, increased secretion of IL12 and IL10 and higher tumour-antigen (TA) uptake capacity. Furthermore, autologus CD3+ T cells responded to TA-loaded mCD40L-activated DC with increased proliferation and cytotoxic response (CD107a and IFN-γ-producing CD3+ CD8+ T cells) to the tumour-loaded autologous PBMCs compared to sCD40L. Thus, these data indicate that mCD40L enhances the immunostimulatory capacity over sCD40L. Furthermore, the ability of mCD40L to also directly induce cell death in CD40-expressing carcinomas, subsequently releasing tumour-specific antigens into the tumour microenvironment highlights the potential for mCD40L as a multi-faceted anti-cancer immunotherapeutic.


Assuntos
Ligante de CD40/metabolismo , Membrana Celular/metabolismo , Linfócitos T/imunologia , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Apoptose/efeitos dos fármacos , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/farmacologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Imunoglobulinas/metabolismo , Interferon gama/metabolismo , Interleucina-10/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Glicoproteínas de Membrana/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
15.
FASEB J ; 34(1): 248-262, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914604

RESUMO

This study was aimed at investigating the therapeutic effects of BITRAP, a bispecific fusion protein targeting TNF-α and IL-21, on the development of autoimmune arthritis in humans and mice. To verify the effects of BITRAP in human, peripheral blood mononuclear cells were cultured with BITRAP under IL-17-producing T (Th17) cell-polarizing conditions or osteoclast differentiation conditions. BITRAP treatment inhibited the production of IL-17 and vascular endothelial growth factor but increased the production of IL-10 in CD4+ T cells, as well as directly suppressed osteoclastogenesis. Collagen-induced arthritis (CIA) and IL-1R antagonist (IL-1Ra) knockout mice were treated with BITRAP. Following injection in CIA mice, BITRAP rapidly migrated into the inflamed joints and remained there for 72 hours. Application of BITRAP attenuated the severity of autoimmune arthritis in CIA and IL-1Ra knockout mice by reducing the numbers of inflammatory cytokine-expressing cells and Th17 cells and antibody secretion. Finally, BITRAP suppressed STAT3 phosphorylation, as well as production of IL-17 and TNF-α, in murine splenic CD4+ T cells. These findings suggest that BITRAP, a bispecific fusion protein targeting TNF-α and IL-21, may be an effective treatment to overcome the limitations of anti-TNF therapy for patients with rheumatoid arthritis.


Assuntos
Artrite/tratamento farmacológico , Interleucinas/antagonistas & inibidores , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Fatores de Coagulação Sanguínea , Linfócitos T CD4-Positivos , Fibroblastos , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Imunoglobulinas/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Osteogênese/efeitos dos fármacos , Engenharia de Proteínas , Proteínas Recombinantes , Células Th17 , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
16.
BMC Biotechnol ; 20(1): 1, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31959159

RESUMO

BACKGROUND: The use of biomaterials has been expanded to improve the characteristics of vaccines. Recently we have identified that the peptide PH(1-110) from polyhedrin self-aggregates and incorporates foreign proteins to form particles. We have proposed that this peptide can be used as an antigen carrying system for vaccines. However, the immune response generated by the antigen fused to the peptide has not been fully characterized. In addition, the adjuvant effect and thermostability of the particles has not been evaluated. RESULTS: In the present study we demonstrate the use of a system developed to generate nano and microparticles carrying as a fusion protein peptides or proteins of interest to be used as vaccines. These particles are purified easily by centrifugation. Immunization of animals with the particles in the absence of adjuvant result in a robust and long-lasting immune response. Proteins contained inside the particles are maintained for over 1 year at ambient temperature, preserving their immunological properties. CONCLUSION: The rapid and efficient production of the particles in addition to the robust immune response they generate position this system as an excellent method for the rapid response against emerging diseases. The thermostability conferred by the particle system facilitates the distribution of the vaccines in developing countries or areas with no electricity.


Assuntos
Antígenos/imunologia , Imunoglobulinas/metabolismo , Proteínas de Matriz de Corpos de Inclusão/química , Peptídeos/química , Vacinas/imunologia , Animais , Antígenos/química , Estabilidade de Medicamentos , Feminino , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/imunologia , Imunização , Camundongos , Nanopartículas , Tamanho da Partícula , Agregados Proteicos , Proteínas Recombinantes de Fusão/imunologia , Termodinâmica , Vacinas/química
17.
Int J Biol Sci ; 16(2): 216-227, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31929750

RESUMO

Background and aims: Dysfunction of the immune regulatory system plays a role in the pathogenesis of allergic rhinitis (AR). The underlying mechanism needs to be further investigated. Published data indicate that soluble CD83 (sCD83) has immune regulatory activities. This study aims to investigate the role of sCD83 in the alleviation of experimental AR. Methods: Peripheral blood samples were obtained from AR patients. Serum levels of sCD83 were determined by enzyme-linked immunosorbent assay. A murine AR model was developed to test the effects of sCD83 on suppressing experimental AR. Results: We found that serum levels of sCD83 in the AR group were lower than that in the healthy control group. A negative correlation was identified between the serum sCD83 levels and the frequency of T helper-2 (Th2) cells. The low serum sCD83 levels were also associated with the Bcl2L12 expression in antigen-specific Th2 cells. Exposure to sCD83 enhanced the responsiveness of antigen-specific Th2 cells to apoptosis inducers via suppressing the Bcl2L12 expression. Administration of sCD83 efficiently suppressed experimental AR. Conclusions: sCD83 contributes to immune homeostasis by regulating CD4+ T cell activities. Administration of sCD83 may have translational potential for the treatment of AR or other allergic diseases.


Assuntos
Antígenos CD/metabolismo , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Rinite Alérgica/metabolismo , Células Th2/metabolismo , Adulto , Animais , Western Blotting , Linfócitos T CD4-Positivos/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hipersensibilidade/metabolismo , Imunoprecipitação , Masculino , Camundongos , Proteínas Musculares/metabolismo , Mucosa Nasal/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pyroglyphidae , Interferência de RNA
18.
Clin Exp Immunol ; 200(1): 73-86, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31859362

RESUMO

B cells orchestrate pro-survival and pro-apoptotic inputs during unfolded protein response (UPR) to translate, fold, sort, secrete and recycle immunoglobulins. In common variable immunodeficiency (CVID) patients, activated B cells are predisposed to an overload of abnormally processed, misfolded immunoglobulins. Using highly accurate transcript measurements, we show that expression of UPR genes and immunoglobulin chains differs qualitatively and quantitatively during the first 4 h of chemically induced UPR in B cells from CVID patients and a healthy subject. We tested thapsigargin or tunicamycin as stressors and 4-phenylbutyrate, dimethyl sulfoxide and tauroursodeoxycholic acid as chemical chaperones. We found an early and robust decrease of the UPR upon endoplasmic reticulum (ER) stress in CVID patient cells compared to the healthy control consistent with the disease phenotype. The chemical chaperones increased the UPR in the CVID patient cells in response to the stressors, suggesting that misfolded immunoglobulins were stabilized. We suggest that the AMP-dependent transcription factor alpha branch of the UPR is disturbed in CVID patients, underlying the observed expression behavior.


Assuntos
Linfócitos B/efeitos dos fármacos , Imunodeficiência de Variável Comum/genética , Dimetil Sulfóxido/farmacologia , Fenilbutiratos/farmacologia , Ácido Tauroquenodesoxicólico/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Imunodeficiência de Variável Comum/metabolismo , Imunodeficiência de Variável Comum/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/imunologia , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Tapsigargina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/genética
19.
Hum Cell ; 33(1): 116-122, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31552567

RESUMO

Human endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2) is a newly identified member of B7 family; HHLA2 protein expression has been suggested to be increased levels in many kinds of human cancers. However, HHLA2 protein expression in gastric cancer tissues and its clinical significance are still unknown. The purpose of this study was to investigate the HHLA2 protein expression pattern in gastric cancer tissues and the correlation between HHLA2 protein expression and clinicopathological characteristics including clinical outcome in gastric cancer patients. In our results, we observed HHLA2 expression was increased in gastric cancer tissue specimens compared with normal stomach tissue specimens through analyzing HHLA2 expression data from 408 gastric cancer tissue specimens and 211 normal stomach tissue specimens at databases. Furthermore, we, respectively, performed quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunohistochemistry to verify HHLA2 mRNA and protein expressions in gastric cancer tissues and normal stomach tissues, and found HHLA2 mRNA and protein expressions were up-regulated in gastric cancer tissues. Moreover, we found high HHLA2 expression was correlated with advanced clinical stage, deep tumor invasion, lymph node metastasis, distant metastasis and short overall survival in gastric cancer. The multivariable Cox's proportional hazard models indicated high HHLA2 expression was a poor independent prognostic factor for overall survival in patients with gastric cancer. In conclusion, HHLA2 protein overexpression in gastric cancer tissue is potential risk factor for malignant status and poor prognosis.


Assuntos
Expressão Gênica , Imunoglobulinas/genética , Neoplasias Gástricas/diagnóstico , Biomarcadores Tumorais/metabolismo , Humanos , Imunoglobulinas/metabolismo , Prognóstico
20.
Biomolecules ; 9(12)2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817821

RESUMO

Post-traumatic stress disorder (PTSD) develops in a portion of individuals exposed to extreme trauma. Glycosylation is a post-translational modification that affects protein functions and is altered in various pathophysiological states and aging. There are still no validated biomarkers of PTSD. The aim of this study was to evaluate the N-glycomic profile in 543 male Caucasian individuals (299 veterans with PTSD and 244 control subjects). The study included discovery (N = 233) and replication (N = 310) cohort. Hydrophilic interaction HPLC and ultra-performance liquid chromatography were used to separate and detect 39 plasma and 24 IgG N-glycan species, respectively. All results were corrected for the effects of age and multiple testing. Significant results included only significantly altered N-glycans in cases/controls in both cohorts, in the same direction. Results showed that six plasma N-glycans (four increased and two decreased) were altered in PTSD vs. controls in both cohorts, but IgG N-glycans were similar between groups. The severity of PTSD was not associated with different plasma N-glycans. This is the first study detecting alterations in plasma N-glycans in PTSD. These N-glycans are also associated with other neuropsychiatric disorders and inflammation, suggesting possible shared glycosylation mechanisms.


Assuntos
Biomarcadores/sangue , Polissacarídeos/sangue , Transtornos de Estresse Pós-Traumáticos/metabolismo , Adulto , Idoso , Biomarcadores/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Glicômica , Glicosilação , Humanos , Imunoglobulinas/sangue , Imunoglobulinas/metabolismo , Inflamação , Masculino , Pessoa de Meia-Idade , Polissacarídeos/metabolismo , Veteranos
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