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1.
Anticancer Res ; 39(9): 4643-4652, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519562

RESUMO

BACKGROUND/AIM: Adenoviral-mediated expression of CD40 ligand (CD40L) on dendritic cells (DCs) activates immune check point CD40/CD40L, enhancing the immunostimulation of DCs and effector cells against human renal carcinoma cells (RCC) and inducing tumor cell apoptosis in vitro. MATERIALS AND METHODS: DCs, isolated from buffy coats from healthy donors, were transduced with adenoviruses carrying human CD40L (Ad-hCD40L). Subsequently maturation marker and cytokine expression were analyzed by fluorescence-activated cell sorting and enzyme-linked immunosorbent assay. RESULTS: Adenoviral transduction induced high expression of soluble CD40L and membrane-bound CD40L, leading to a strong CD40-CD40L interaction in DCs. Interestingly, a T-helper cell type 1 shift of expressed cytokines/chemokines was observed due to the expression of membrane-bound CD40L rather than due to soIuble CD40L alone, which significantly reduced immunoactivation of DCs. However, supernatants of Ad-hCD40L-transduced DCs induced apoptosis of RCC cells. Co-culture of Ad-hCD40L DCs with cytokine-induced killer cells led to a significant stimulation of tumor-specific cytokine-induced killer cells, with increased proliferation and cytotoxicity. CONCLUSION: Use of Ad-hCD40L-transduced DCs is a promising approach to treating RCC.


Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Carcinoma de Células Renais/metabolismo , Células Dendríticas/metabolismo , Imunomodulação , Neoplasias Renais/metabolismo , Adenoviridae/genética , Biomarcadores , Ligante de CD40/genética , Carcinoma de Células Renais/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Neoplasias Renais/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transdução Genética
2.
Scand J Immunol ; 90(4): e12798, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31179555

RESUMO

CD40 ligand (CD40L) deficiency is a rare but life-threatening primary immunodeficiency caused by mutations in the CD40L gene. Here, we investigated a cohort of 40 genetically diagnosed CD40L-deficient patients from the Chinese mainland, analysed their clinical and genetic data, and examined CD40L expression, the proportion of T cell subsets, B cell subsets and T follicular helper (Tfh) cells. The aim was to provide a complete picture of CD40L deficiency. Initial presentations of the patient cohort mainly involved recurrent fever (47.5%) and sinopulmonary infection (42.5%). Life-threatening infections (42.5%), caused by various pathogens, were the most serious threats faced by CD40L-deficient patients, while neutropenia (57.5%) remained the most common complication. Opportunistic infections, including Pneumocystis carinii pneumonia and invasive fungal disease associated with Talaromyces marneffei, were also common in the cohort. In addition, seven patients (17.5%) suffered BCGitis/BCGosis, which is a major problem facing a planned immunization programme in China. It was intriguing that reduced IgM levels were observed in 12.5% of patients, while normal or elevated IgA levels were shown in 47.5% of patients. Thirty-seven unique mutations were identified in 40 patients; of these, 10 were novel. Furthermore, we observed a lower percentage of NK cells, Tfh cells, and central memory CD4+ T cells, and an extremely small class-switched memory B cell population, in CD40L-deficient patients. Patients who underwent hematopoietic stem cell transplantation experienced better disease remission. Taken together, our data establish the largest database about CD40L deficiency in China and provide genetic, immunologic and clinical information about Chinese CD40L-deficient patients.


Assuntos
Ligante de CD40/genética , Síndromes de Imunodeficiência/imunologia , Células Matadoras Naturais/imunologia , Pneumopatias Fúngicas/imunologia , Pneumocystis carinii/fisiologia , Pneumonia por Pneumocystis/imunologia , Linfócitos T/imunologia , Talaromyces/fisiologia , China , Estudos de Coortes , Febre , Humanos , Imunoglobulina M/sangue , Síndromes de Imunodeficiência/genética , Memória Imunológica , Pneumopatias Fúngicas/genética , Masculino , Mycobacterium bovis , Pneumonia por Pneumocystis/genética , Adulto Jovem
3.
Scand J Immunol ; 90(2): e12776, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31069824

RESUMO

The number of the X chromosome-linked genes has been previously suggested to influence immune responses and the development of autoimmune diseases. In the present study, we aimed at evaluating the level of expression of CD40L (an X-linked gene involved in adaptive immunity) and TLR7 (an X-linked gene involved in innate immunity) in a variety of different karyotypes. Those included males, females and patients with X chromosome aneuploidy. Healthy females (46, XX; n = 10) and healthy males (46, XY; n = 10) were compared to females with Turner syndrome (TS) (45, X; n = 11) and males with Klinefelter syndrome (KS) (47, XXY; n = 5). Stimulation of peripheral blood mononuclear cells (PBMCs) with PMA and ionomycin resulted in higher percentage of CD3 + CD40L+ T cells (P < 0.001) and higher level expression of CD40L in T cell (P < 0.001) in female and KS patients compared with male and TS patients. TLR7-mediated IFN-alpha production by HLADR + CD3- CD19- cells was significantly upregulated in healthy women compared with healthy males, TS and KS patients (P < 0.001). TLR7 agonist-stimulated PBMCs from healthy females and KS patients expressed significantly higher levels of TLR7 mRNA than those from male and TS patients (P < 0.05). The increased expression of the X-linked genes TLR7 and CD40L in healthy females and KS patients suggests that the presence of two X chromosomes plays a major role in enhancing both innate and adaptive immune responses. These results may contribute to the explanation of sex-based differences in immune biology and the sex bias in predisposition to autoimmune diseases.


Assuntos
Imunidade Adaptativa/genética , Ligante de CD40/biossíntese , Ligante de CD40/genética , Cromossomos Humanos X/genética , Dosagem de Genes/genética , Imunidade Inata/genética , Receptor 7 Toll-Like/biossíntese , Receptor 7 Toll-Like/genética , Imunidade Adaptativa/imunologia , Antígenos CD19/biossíntese , Complexo CD3/biossíntese , Células Cultivadas , Variações do Número de Cópias de DNA/genética , Feminino , Humanos , Imunidade Inata/imunologia , Interferon-alfa/biossíntese , Ionomicina/farmacologia , Síndrome de Klinefelter/genética , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Ácidos Polimetacrílicos/farmacologia , RNA Mensageiro/biossíntese , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Síndrome de Turner/genética
4.
J Med Microbiol ; 68(3): 480-492, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30657443

RESUMO

PURPOSE: Hantavirus infections cause severe haemorrhagic fever with renal syndrome (HFRS) in humans and are associated with high fatality rates. In 2017, numerous outbreaks were reported in China and Germany. This represents a significant public-healthcare issue with no effective HFRS vaccines that offer a long-term immune response. In this study, we investigated the long-term humoral and cellular immune responses and protective immunity of Hantaan virus (HTNV) granulocyte-macrophage colony stimulating factor (GM-CSF) and CD40 ligand (CD40L) virus-like particles (VLPs) in mice. METHODOLOGY: GM-CSF and CD40L VLPs were constructed via co-transfection of pCI-S and pCI-M-CD40L, and pCI-S and pCI-M-GM-CSF, into dihydrofolatereductase (dhfr)-deficient Chinese hamster ovary cells, respectively. Mice were immunized with HTNV VLPs 2 weeks apart. The animals were challenged 6 months after immunization. Specific and neutralizing antibodies were assessed by ELISA; IFN-γ was measured by enzyme-linked immunospot (ELISpot) assay and effectiveness by cytotoxic T lymphocyte (CTL) cytotoxicity assays. Nucleic acid loads of HTNV were tested by quantitative real-time PCR and viral antigen was detected via indirect ELISA. Pathological alterations were detected via haematoxylin-eosin staining. RESULTS: GM-CSF and CD40L VLPs provided stable, long-term protection with a high titre of neutralizing antibody in mice 6 months after immunization. Furthermore, VLPs increased HTNV-specific cellular immune responses via higher expression of IFN-γ and CTL responses. HTNV challenge assay results showed long-term protection against HFRS. No significant pathological alteration was observed in the organs of mice after immunization. CONCLUSION: This is, to the best of our knowledge, the first report demonstrating the long-term potency of HTNV VLP vaccines against HTNV infection and offers new insights into HTNV vaccine development.


Assuntos
Ligante de CD40/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Infecções por Hantavirus/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Ligante de CD40/administração & dosagem , Células CHO , Cricetulus , Ensaio de Imunoadsorção Enzimática , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Vírus Hantaan/genética , Infecções por Hantavirus/imunologia , Imunidade Celular , Imunidade Humoral , Camundongos , Camundongos Endogâmicos C57BL , Transfecção , Vacinas de Partículas Semelhantes a Vírus/genética
5.
J Immunol ; 202(3): 787-798, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30617223

RESUMO

IL-6 plays a role in cancer pathogenesis via its connection to proteins involved in the formation of desmoplastic stroma and to immunosuppression by driving differentiation of myeloid suppressor cells together with TGF-ß. Inhibition of IL-6 signaling in the tumor microenvironment may, thus, limit desmoplasia and myeloid suppressor cell differentiation. CD40 signaling can further revert myeloid cell differentiation toward antitumor active phenotypes. Hence, the simultaneous use of IL-6 blockade with CD40 stimuli may tilt the tumor microenvironment to promote antitumor immune responses. In this paper, we evaluated the mechanisms of LOAd713, an oncolytic adenovirus designed to block IL-6R signaling and to provide myeloid cell activation via a trimerized membrane-bound isoleucine zipper (TMZ) CD40L. LOAd713-infected pancreatic cancer cells were killed by oncolysis, whereas infection of stellate cells reduced factors involved in stroma formation, including TGF-ß-1 and collagen type I. Virus infection prevented IL-6/GM-CSF-mediated differentiation of myeloid suppressors, but not CD163 macrophages, whereas infection of dendritic cells led to upregulation of maturation markers, including CD83, CD86, IL-12p70, and IFN-γ. Further, IL-6R blockade prevented upregulation of programed death ligand 1 (PD-L1) and PD-1 on the stimulated dendritic cells. These results suggest that LOAd713 can kill infected tumor cells and has the capacity to affect the tumor microenvironment by stimulating stellate cells and myeloid suppressors with TMZ-CD40L and IL-6R blockade. Gene transfer of murine TMZ-CD40L prolonged survival in an animal model. LOAd713 may be an interesting therapeutic option for cancers connected to IL-6 signaling, such as pancreatic cancer.


Assuntos
Ligante de CD40/metabolismo , Ativação Linfocitária , Terapia Viral Oncolítica , Receptores de Interleucina-6/antagonistas & inibidores , Transdução de Sinais , Adenoviridae , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Ligante de CD40/genética , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Células Dendríticas/citologia , Terapia Genética , Vetores Genéticos , Células HEK293 , Humanos , Interleucina-6/imunologia , Zíper de Leucina , Melanoma Experimental , Camundongos , Neoplasias Pancreáticas/imunologia , Células Estreladas do Pâncreas/citologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Receptores de Interleucina-6/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral/imunologia
6.
Mol Immunol ; 105: 173-180, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30529036

RESUMO

CD40 ligand (CD40 L) expressed by activated T cells interacts with CD40 on B cells and triggers B cell survival, proliferation and differentiation. Deficiency in CD40 L or CD40 in humans causes hyper IgM syndrome due to a defect in T-B interaction that is essential for Ig gene class switch recombination (CSR). CD40 L belongs to the tumor necrosis factor family and normally forms a homotrimer on the cell surface, which is important for its biological activity. To generate a multimeric CD40 L that can be used to stimulate both mouse and human B cells, we fused the extracellular domain of mouse CD40 L, which is known to also bind human CD40, with streptavidin (SA) that forms a stable tetramer under physiological conditions. As expected, 293 T cells transiently transfected with an SA-CD40 L expression vector secreted tetrameric SA-CD40 L in the culture supernatant. The secreted SA-CD40 L exhibited > 25-fold stronger activities in inducing the survival, activation and proliferation of both mouse and human primary B cells than did an agonistic anti-mouse or anti-human CD40 antibody. In the presence of IL-4, SA-CD40 L also induced efficient CSR and plasma cell differentiation in both mouse and human B cells. Moreover, administration of SA-CD40 L in mice induced activation and proliferation of spleen B cells in vivo. These results demonstrate that the SA-CD40 L fusion protein generated in the present study recapitulates the function of membrane-bound trimeric CD40 L and has potent biological activities in both mouse and human primary B cells.


Assuntos
Ligante de CD40/farmacologia , Diferenciação Celular/efeitos dos fármacos , Plasmócitos/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Antígenos CD40/imunologia , Ligante de CD40/genética , Ligante de CD40/imunologia , Diferenciação Celular/imunologia , Feminino , Humanos , Síndrome de Imunodeficiência com Hiper-IgM/tratamento farmacológico , Síndrome de Imunodeficiência com Hiper-IgM/genética , Síndrome de Imunodeficiência com Hiper-IgM/imunologia , Síndrome de Imunodeficiência com Hiper-IgM/patologia , Masculino , Camundongos , Plasmócitos/patologia , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia
7.
Cell Physiol Biochem ; 48(4): 1793-1803, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30078020

RESUMO

BACKGROUND/AIMS: Cellular senescence acts as a barrier against tumorigenesis. The CD40L transgene, expressed in some tumor cells, not only becomes visible to antigen-presenting cells but also actively catalyzes its own termination. Here, we evaluated the effect of a membrane-bound mutant form of human CD40L (CD40L-M) on senescence and the senescence-associated secretory phenotype (SASP) in non-small cell lung cancer (NSCLC). METHODS: CD40 expression levels in the NSCLC cell lines A549/TR, A549/DDP and H460 were examined by flow cytometry. Senescent cells and tissues were identified via SA-ß-gal activity. Cell proliferation was visualized by EdU labeling. qRT-PCR, Western blotting, and immunofluorescence staining were conducted to assess mRNA and protein expression levels of CD40L, γ-H2A.X, p65, p-p65, IκBα, p53, p21 and p16. Cytokines secreted from transfected cells were tested by ELISA and cell migration assay. Capsid tyrosine-modified rAAV5-CD40L-M was packaged and carried out in vivo. RESULTS: Overexpression of CD40L-M promoted senescence, inhibited proliferation, increased DNA damage-associated γ-H2A.X, and initiated the SASP in CD40-positive NSCLC cells. NF-κB signaling was activated by CD40L-M overexpression in these cells. Knockdown of NF-κB partially overcame senescence and failed to induce SASP. Furthermore, increased p53 and p21 protein levels induced by CD40L-M were also reduced following NF-κB suppression. CONCLUSIONS: These data showed that the membrane-bound CD40L mutant may promote cellular senescence and initiate the SASP of NSCLC cells in an NF-κB-dependent manner. Therefore, CD40L-M-induced senescence may be a potential approach to protect against lung adenocarcinoma.


Assuntos
Adenocarcinoma/patologia , Ligante de CD40/metabolismo , Neoplasias Pulmonares/patologia , NF-kappa B/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Animais , Antineoplásicos Fitogênicos/farmacologia , Ligante de CD40/antagonistas & inibidores , Ligante de CD40/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Histonas/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Paclitaxel/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/metabolismo
8.
Biomed Res Int ; 2018: 7497314, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29780830

RESUMO

Sepsis is a severe and progressive disease characterized by systemic inflammatory response syndrome (SIRS). CD40 serves as a vital link between immune response and inflammation. This study was designed to investigate the potential association between a functional single-nucleotide polymorphism (SNP) of CD40 (rs1883832) and susceptibility to sepsis. We first performed a case-control study to explore the relationship between the CD40 rs1883832 polymorphism and sepsis. CD40 mRNA expression and protein expression were determined by real-time PCR and western blotting, respectively, in peripheral blood mononuclear cells (PBMCs) from sepsis patients and healthy controls. The plasma sCD40L levels in the two groups were measured by ELISA. The results showed that the frequencies of the TT genotype and the CD40 rs1883832 T allele were significantly higher in sepsis patients than in healthy controls. Plasma sCD40L levels were also significantly increased in sepsis patients. In addition, TT genotype carriers among sepsis patients displayed the highest CD40 expression at both the mRNA and protein levels, accompanied by the highest plasma sCD40L concentrations. In conclusion, the CD40 rs1883832 T allele acts as a risk factor for increased susceptibility to sepsis and may be involved in the process of sepsis through regulation of CD40 expression and plasma sCD40L levels.


Assuntos
Antígenos CD40 , Ligante de CD40 , Regulação da Expressão Gênica , Predisposição Genética para Doença , Polimorfismo Genético , Sepse , Adulto , Idoso , Grupo com Ancestrais do Continente Asiático , Antígenos CD40/sangue , Antígenos CD40/genética , Ligante de CD40/sangue , Ligante de CD40/genética , China , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Sepse/sangue , Sepse/genética
9.
Indian J Pathol Microbiol ; 61(1): 137-140, 2018 Jan-Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29567905

RESUMO

X-linked hyperimmunoglobulin M (HIGM) syndrome may increase the susceptibility of patients to disseminated cryptococcal infections primarily due to CD40L deficiency that causes defective cross talk between T- and B-cells, thus preventing class switching. In HIGM syndrome, serum IgM levels are elevated with severe reduction in serum immunoglobulin G (IgG) and IgA levels. In addition, the expression of CD40L (CD154) on in vitro-activated T-cells is severely reduced or absent. Here, we describe a rare, and perhaps, the first reported case in India of a 3-year-old male child with X-linked HIGM immunodeficiency syndrome who developed disseminated Cryptococcosis. Evaluation of the serum IgG profile of the patient revealed increased serum IgM levels with reduced IgG and IgA levels. Both the frequency and the function of T-cells, primarily CD40L on activated T-cells, showed weak expression suggestive of HIGM syndrome.


Assuntos
Ligante de CD40/genética , Criptococose/sangue , Criptococose/imunologia , Linfócitos T/imunologia , Linfócitos B/imunologia , Ligante de CD40/imunologia , Pré-Escolar , Criptococose/diagnóstico , Criptococose/epidemiologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Síndromes de Imunodeficiência/congênito , Índia/epidemiologia , Masculino , Mutação
10.
Rheumatology (Oxford) ; 57(6): 1105-1114, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29522194

RESUMO

Objective: Immunosuppressive biologics are used in the management of RA and additional immunomodulators are under investigation including modulators of the CD40/CD40 ligand (CD40L) costimulation pathway. Tampering with immune function can have unanticipated skeletal consequences due to disruption of the immuno-skeletal interface, a nexus of shared cells and cytokine effectors serving discrete functions in both immune and skeletal systems. In this study, we examined the effect of MR1, a CD40L neutralizing antibody, on physiological bone remodelling in healthy mice. Methods: Female C57BL6 mice were treated with MR1 and BMD was quantified by dual energy X-ray absorptiometry and indices of trabecular bone structure were quantified by micro-CT. Serum biochemical markers were used to evaluate bone turnover and formation indices by histomorphometry. Results: Unexpectedly, MR1 stimulated significant accretion of BMD and trabecular bone mass in the spine, but not in long bones. Surprisingly, bone accretion was accompanied by a significant increase in bone formation, rather than suppression of bone resorption. Mechanistically, MR1-induced bone accrual was associated with increased Treg development and elevated production of cytotoxic T lymphocyte antigen 4, a costimulation inhibitor that promotes T cell anergy and CD8+ T cell expression of the bone anabolic ligand Wnt-10b. Conclusion: Our studies reveal an unexpected bone anabolic activity of pharmacological CD40L suppression. Therapeutic targeting of the CD40L pathway may indeed have unforeseen consequences for the skeleton, but may also constitute a novel strategy to promote bone formation to ameliorate osteoporotic bone loss and reduce fracture risk in the axial skeleton.


Assuntos
Artrite Reumatoide/genética , Ligante de CD40/genética , Osso Esponjoso/metabolismo , Regulação da Expressão Gênica , Vértebras Lombares/metabolismo , Osteogênese/genética , Absorciometria de Fóton , Animais , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/metabolismo , Ligante de CD40/biossíntese , Ligante de CD40/imunologia , Osso Esponjoso/diagnóstico por imagem , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Vértebras Lombares/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos C57BL , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Microtomografia por Raio-X
11.
Oncol Rep ; 39(5): 2071-2080, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29565449

RESUMO

The ligand of CD40, known as CD154 or CD40L, is the key to immunostimulatory and anticancer activity, but how CD40L affects cellular senescence is unclear. Thus, we studied a membrane­stable mutant form CD40L (CD40L­M) to explore tumor growth and cellular senescence in CD40­positive NSCLC cells. We found that CD40L­M­expressing cells had senescent characteristics, including reduced cell proliferation and enlargement, increased SA­ß­gal staining activity, and overexpression of several cell cycle regulators p53 and p21. In addition, expression of GATA4 was restored, and the NF­κB signaling pathway was activated in the CD40L­M­induced senescent cells. Mechanistic analyses revealed that CD40L­M expression triggered the ATM/Chk2 DNA damage response, which mediated cell senescence and GATA4 activation. Knockdown of GATA4 reversed CD40L­M­induced senescence and decreased NF­κB activity. Thus, CD40L­M contributes to induction of cell senescence in CD40­positive NSCLC cells, and GATA4 is a switch to activate the NF­κB pathway, which is positively regulated by DNA damage response (DDR) signaling kinases. Collectively, CD40L­M­induced senescence may be a barrier to the growth of lung cancer cells.


Assuntos
Ligante de CD40/genética , Dano ao DNA , Fator de Transcrição GATA4/genética , Neoplasias Pulmonares/genética , Mutação , Células A549 , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
12.
Biologicals ; 52: 67-71, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29311002

RESUMO

The development of subunit vaccines against classical swine fever is a desirable goal, because it allows discrimination between vaccinated and infected animals. In this study, humoral and cellular immune response elicited in inbred BALB/c mice by immunization with a recombinant classical swine fever virus (CSFV) E2 protein fused to porcine CD154 antigen (E2CD154) was assessed. This model was used as a predictor of immune response in swine. Mice were immunized with E2CD154 emulsified in Montanide ISA50V2 or dissolved in saline on days 1 and 21. Another group received E2His antigen, without CD154, in the same adjuvant. Montanide ISA50V2 or saline served as negative controls for each experimental group. Animals immunized with 12.5 and 2.5 µg/dose of E2CD154 developed the highest titers (>1:2000) of CSFV neutralizing antibodies. Moreover, CSFV specific splenocyte gamma-interferon production, measured after seven and twenty-eight days of immunization, was significantly higher in mice immunized with 12.5 µg of E2CD154. As a conclusion, E2CD154 emulsified in Montanide ISA50 V2 was able to induce a potent humoral and an early cellular immune response in inbred BALB/c mice. Therefore, this immunogen might be an appropriate candidate to elicit immune response in swine, control CSF disease and to eliminate CSFV in swine.


Assuntos
Ligante de CD40/imunologia , Imunidade Celular , Imunidade Humoral , Imunogenicidade da Vacina , Proteínas Recombinantes de Fusão/imunologia , Proteínas Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ligante de CD40/genética , Vírus da Febre Suína Clássica , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Suínos , Proteínas Virais/genética , Vacinas Virais/genética
13.
Protein Expr Purif ; 142: 37-44, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28974444

RESUMO

Delivering antigen via molecules specifically targeting receptors on the surface of antigen-presenting cells is a strategy to improve immune responses. In this study, an antigen-targeting fusion protein (OVA-CD40LS) composed of the C-terminal fragment of ovalbumin and the extracellular domain of mouse CD40 ligand was constructed by genetic fusion. The OVA-CD40LS and the control OVA (rOVA) genes were cloned in Escherichia coli and over-expressed as insoluble proteins. The rOVA protein was purified from the insoluble fraction of E. coli cell lysate by nickel affinity chromatography and refolded by step-wise dialysis to give a yield of 11.8 mg/L of culture. The OVA-CD40LS was purified by a 'two-round' nickel affinity and on-column protein-refolding chromatography. The yield was 528 µg/L of culture. The purified OVA-CD40LS, but not the rOVA, was able to simulate the production of pro-inflammatory cytokines and up-regulate cell surface marker proteins in mouse bone marrow-derived dendritic cells. The purified OVA-CD40LS elicited a robust immune response when injected submucosally in the oral cavity of mice. Collectively, the results indicate that the OVA-CD40LS fusion protein was biologically active, functioning as an antigen-targeting protein.


Assuntos
Ligante de CD40/imunologia , Células Dendríticas/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Ovalbumina/imunologia , Plasmídeos/química , Proteínas Recombinantes de Fusão/biossíntese , Animais , Anticorpos/sangue , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Ligante de CD40/genética , Clonagem Molecular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Imunização , Interleucina-6/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Bucal/citologia , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/imunologia , Ovalbumina/genética , Plasmídeos/metabolismo , Cultura Primária de Células , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Fator de Necrose Tumoral alfa/biossíntese
14.
Arch Virol ; 163(4): 855-865, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29248968

RESUMO

We found a HLA class II histocompatibility antigen gene, DQ alpha 1 chain (HLA-DQA1), that was expressed more than 9-fold higher in high-load hepatitis C virus (HCV) livers than low-load HCV livers using transcriptomics of chronic HCV-infected livers. To further investigate this finding, we examined which cells were positive for HLA-DQA1 and what liver immune responses were different between HCV-high and -low livers. HLA-DQA1-positive cells were significantly increased in the HCV-high group, and most positive cells were identified as non-parenchymal sinusoid cells and lymphocytic infiltrates in the portal area. Parenchymal hepatocytes were negative for HLA-DQA1. HLA-DQA1-positive cells in the liver sinusoid were positive for CD68 (macrophages or Kupffer cells); those in the lymphocytic infiltrates were positive for CD20 (B cells) or CD3 (T cells). mRNA levels of antigen-presenting cell (APC) markers such as CD68 and CD11c were significantly upregulated in the HCV-high group and were correlated with HLA-DQA mRNA levels. CD8B mRNA (CD8+ T cells) was upregulated in both HCV-positive livers compared with HCV-negative livers, whereas CD154 mRNA (CD4+ T helper cell) was upregulated in the HCV-high group compared with the HCV-low group. The immune regulatory molecules FOXP3 mRNA (regulatory T cell, T reg) and programmed cell death ligand-1 (PD-L1) mRNA were significantly increased in the HCV-high group. HCV-high livers had two molecular immune responses: increased APC numbers and adaptive immunity and the induction of immune tolerance. The local hepatic imbalance of contradictory immune responses might be responsible for high HCV loads.


Assuntos
Carcinoma Hepatocelular/genética , Cadeias alfa de HLA-DQ/genética , Hepatite C Crônica/genética , Neoplasias Hepáticas/genética , Carga Viral/genética , Imunidade Adaptativa , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD20/genética , Antígenos CD20/imunologia , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/imunologia , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Ligante de CD40/genética , Ligante de CD40/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/virologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica , Cadeias alfa de HLA-DQ/imunologia , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/imunologia , Hepacivirus/patogenicidade , Hepatite C Crônica/complicações , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Tolerância Imunológica , Macrófagos do Fígado/imunologia , Macrófagos do Fígado/virologia , Fígado/imunologia , Fígado/virologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/virologia , Transdução de Sinais , Transcriptoma/imunologia , Carga Viral/imunologia
15.
Clin Rheumatol ; 37(2): 345-353, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28963582

RESUMO

The objective of this study was to determine the association of the CD40LG 3'-UTR (CA)n microsatellite with rheumatoid arthritis (RA) and CD40LG mRNA levels in females from western Mexico. A case-control study with 219 RA patients and 175 control subjects (CS) was conducted. Genotyping was performed by polymerase chain reaction (PCR), X 2 test was used to compare genotype and allele frequencies, and odds ratios and 95% confidence intervals were calculated to evaluate the association between RA and the microsatellite. CD40LG mRNA expression was assessed by real-time quantitative PCR. For comparisons between groups, Kruskal-Wallis or Mann-Whitney U tests for non-parametric data and ANOVA test for parametric data were performed. Among the 13 different alleles identified, CA25 was the most represented (45.4% RA and 46.3% CS). Stratification according to CA repeats as CA25 showed a tendency towards a higher frequency of >CA25 alleles in RA patients (29%) compared to CS (23.4%). There was no association between any genotype and the clinical parameters of RA patients. According to the 2-∆∆Cq method, CD40LG mRNA expression in RA patients was 4.5-fold higher compared to CS; this difference was significant when assessed by the 2-∆Cq method (p = 0.028). Compared to carriers of the CA25/CA25 genotype, CS carrying the CA25/>CA25 carriers. The 3'-UTR CD40LG (CA)n microsatellite is not a genetic marker for RA in western Mexican population; however, results suggest that it plays a role in the CD40LG mRNA expression.


Assuntos
Regiões 3' não Traduzidas , Artrite Reumatoide/diagnóstico , Ligante de CD40/genética , Marcadores Genéticos , Repetições de Microssatélites , Adulto , Alelos , Artrite Reumatoide/genética , Estudos de Casos e Controles , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , México , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética
16.
Proc Natl Acad Sci U S A ; 114(48): 12797-12802, 2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-29133396

RESUMO

Follicular helper T cells (TFHs) are a key component of adaptive immune responses as they help antibody production by B cells. Differentiation and function of TFH cells are controlled by the master gene BCL6, but it is largely unclear how this transcription repressor specifies the TFH program. Here we asked whether BCL6 controlled helper function through down-regulation of specific microRNAs (miRNAs). We first assessed miRNA expression in TFH cells and defined a TFH-specific miRNA signature. We report that hsa-miR-31-5p (miR-31) is down-regulated in TFH; we showed that BCL6 suppresses miR-31 expression by binding to its promoter; and we demonstrated that miR-31 inhibits the expression of molecules that control T-helper function, such as CD40L and SAP. These findings identify a BCL6-initiated inhibitory circuit that stabilizes the follicular helper T cell program at least in part through the control of miRNA transcription. Although BCL6 controls TFH activity in human and mouse, the role of miR-31 is restricted to human TFH cell differentiation, reflecting a species specificity of the miR-31 action. Our findings highlight miR-31 as a possible target to modulate human T cell dependent antibody responses in the settings of infection, vaccination, or immune dysregulation.


Assuntos
Linfócitos B/imunologia , Ligante de CD40/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/genética , Linfócitos T Auxiliares-Indutores/imunologia , Imunidade Adaptativa , Animais , Linfócitos B/citologia , Ligante de CD40/imunologia , Diferenciação Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Centro Germinativo/citologia , Centro Germinativo/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/imunologia , Cultura Primária de Células , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Transdução de Sinais , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/imunologia , Especificidade da Espécie , Linfócitos T Auxiliares-Indutores/citologia
17.
Nat Commun ; 8(1): 1447, 2017 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-29129918

RESUMO

CD40 agonists bind the CD40 molecule on antigen-presenting cells and activate them to prime tumor-specific CD8+ T cell responses. Here, we study the antitumor activity and mechanism of action of a nonreplicating adenovirus encoding a chimeric, membrane-bound CD40 ligand (ISF35). Intratumoral administration of ISF35 in subcutaneous B16 melanomas generates tumor-specific, CD8+ T cells that express PD-1 and suppress tumor growth. Combination therapy of ISF35 with systemic anti-PD-1 generates greater antitumor activity than each respective monotherapy. Triple combination of ISF35, anti-PD-1, and anti-CTLA-4 results in complete eradication of injected and noninjected subcutaneous tumors, as well as melanoma tumors in the brain. Therapeutic efficacy is associated with increases in the systemic level of tumor-specific CD8+ T cells, and an increased ratio of intratumoral CD8+ T cells to CD4+ Tregs. These results provide a proof of concept of systemic antitumor activity after intratumoral CD40 triggering with ISF35 in combination with checkpoint blockade for multifocal cancer, including the brain.


Assuntos
Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Antígenos CD40/agonistas , Ligante de CD40/imunologia , Linfócitos T CD8-Positivos/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Adenoviridae/genética , Animais , Encéfalo/patologia , Relação CD4-CD8 , Antígenos CD40/metabolismo , Ligante de CD40/genética , Antígeno CTLA-4/antagonistas & inibidores , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/biossíntese
18.
PLoS One ; 12(11): e0188045, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29155846

RESUMO

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.


Assuntos
Antígenos CD40/genética , Ligante de CD40/genética , Glomerulonefrite Membranosa/metabolismo , Glomerulosclerose Segmentar e Focal/metabolismo , Glomérulos Renais/metabolismo , Síndrome Nefrótica/metabolismo , Adolescente , Corticosteroides/uso terapêutico , Adulto , Albuminas/genética , Albuminas/metabolismo , Animais , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Ligante de CD40/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/patologia , Permeabilidade da Membrana Celular , Criança , Pré-Escolar , Citotoxinas/uso terapêutico , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica , Glomerulonefrite Membranosa/tratamento farmacológico , Glomerulonefrite Membranosa/genética , Glomerulonefrite Membranosa/patologia , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/cirurgia , Soluções para Hemodiálise/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Transplante de Rim , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/genética , Síndrome Nefrótica/patologia , Troca Plasmática , Plasmaferese , Ratos
19.
PLoS One ; 12(10): e0186573, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29023539

RESUMO

Upon recognition of peptide displayed on MHC molecules, Th1 and Th2 cells form distinct immunological synapse structures. Th1 cells have a bull's eye synapse structure with TCR/ MHC-peptide interactions occurring central to a ring of adhesion molecules, while Th2 cells have a multifocal synapse with small clusters of TCR/MHC interactions throughout the area of T cell/antigen-presenting cell interaction. In this study, we investigated whether this structural difference in the immunological synapse affects delivery of T cell help. The immunological synapse is thought to ensure antigen-specific delivery of cytolytic granules and killing of target cells by NK cells and cytolytic T cells. In helper T cells, it has been proposed that the immunological synapse may direct delivery of other effector molecules including cytokines. CD40 ligand (CD40L) is a membrane-bound cytokine essential for antigen-specific T cell help for B cells in the antibody response. We incubated Th1 and Th2 cells overnight with a mixture of antigen-presenting and bystander B cells, and the delivery of CD40L to B cells and subsequent B cell responses were compared. Despite distinct immunological synapse structures, Th1 and Th2 cell do not differ in their ability to deliver CD40L and T cell help in an antigen-specific fashion, or in their susceptibility to inhibition of help by a blocking anti-CD40L antibody.


Assuntos
Células Apresentadoras de Antígenos/metabolismo , Linfócitos B/metabolismo , Ligante de CD40/metabolismo , Sinapses/química , Células Th2/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Células Apresentadoras de Antígenos/citologia , Linfócitos B/imunologia , Antígenos CD40/deficiência , Antígenos CD40/genética , Ligante de CD40/genética , Ligante de CD40/imunologia , Ciclosporina/farmacologia , Feminino , Sinapses Imunológicas/química , Sinapses Imunológicas/efeitos dos fármacos , Sinapses Imunológicas/imunologia , Interleucina-4/imunologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Sinapses/metabolismo , Células Th1/citologia , Células Th1/metabolismo , Células Th2/citologia
20.
Am J Physiol Lung Cell Mol Physiol ; 313(6): L973-L990, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-28912381

RESUMO

Our previous study showed that invariant natural killer T (iNKT) cells might act as an adjuvant to promote Th2 inflammatory responses in an OVA-induced mouse model of allergic asthma, but the mechanism remains unknown. To clarify the underlying mechanism through which iNKT cells promote Th2 inflammatory responses, we investigated the modulatory influence of iNKT cells on phenotypic and functional maturation of lung dendritic cells (LDCs) using iNKT cell-knockout mice, specific iNKT cell activation, coculture experiments, and adoptive transfer of iNKT cells in mouse models of asthma. Our data showed that iNKT cell deficiency could downregulate surface maturation markers and proinflammatory cytokine secretion of LDCs from a mouse model of asthma. However, elevated activation of iNKT cells by α-galactosylceramide and adoptive transfer of iNKT cells could upregulate surface maturation markers and proinflammatory cytokine secretion of LDCs from mouse models of asthma. Meanwhile, iNKT cells significantly influenced the function of LDCs, markedly enhancing Th2 responses in vivo and in vitro. In addition, iNKT cell can induce LDCs expression of CD206 and RELM-α, reflecting alternative activation of LDCs in a mouse model of asthma. α-Galactosylceramide treatment significantly enhanced expression of CD40L of lung iNKT cells from a mouse model of asthma, and the coculture experiment of LDCs with iNKT cells showed that the blockade of CD40L strongly suppressed surface maturation markers and proinflammatory cytokine production by LDCs. Our data suggest that iNKT cells can promote immunogenic maturation of LDCs to enhance Th2 responses in mouse models of asthma.


Assuntos
Asma/imunologia , Células Dendríticas/imunologia , Pulmão/imunologia , Células T Matadoras Naturais/imunologia , Células Th2/imunologia , Animais , Asma/genética , Asma/patologia , Ligante de CD40/genética , Ligante de CD40/imunologia , Células Dendríticas/patologia , Modelos Animais de Doenças , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células T Matadoras Naturais/patologia , Células Th2/patologia
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