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1.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31527122

RESUMO

We previously demonstrated that recombinant protein PAc could be administered as an anticaries vaccine. However, the relatively weak immunogenicity of PAc limits its application. In the present study, we investigated the effect of two adjuvant combinations of chitosan plus Pam3CSK4 (chitosan-Pam3CSK4) and of chitosan plus monophosphoryl lipid A (chitosan-MPL) in the immune responses to the PAc protein in vivo and in vitro PAc-chitosan-Pam3CSK4 or PAc-chitosan-MPL promoted significantly higher PAc-specific antibody titers in serum and saliva, inhibited Streptococcus mutans colonization onto the tooth surfaces, and endowed better protection effect with significantly less caries activities than PAc alone. Chitosan-Pam3CSK4 and chitosan-MPL showed no statistically significant differences. In conclusion, our study demonstrated that the chitosan-Pam3CSK4 and chitosan-MPL combinations are promising for anticaries vaccine development.


Assuntos
Vacinas Bacterianas/imunologia , Quitosana/farmacologia , Cárie Dentária/prevenção & controle , Lipídeo A/análogos & derivados , Lipopeptídeos/farmacologia , Streptococcus mutans/imunologia , Adjuvantes Imunológicos , Animais , Cárie Dentária/microbiologia , Feminino , Imunogenicidade da Vacina/imunologia , Imunoglobulina A Secretora/análise , Lipídeo A/imunologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas NLR/agonistas , Streptococcus mutans/patogenicidade , Receptores Toll-Like/agonistas , Vacinas Sintéticas/imunologia , Fatores de Virulência/imunologia
2.
World J Gastroenterol ; 25(29): 3920-3928, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31413527

RESUMO

In contrast to other tumor types, immunotherapy has not yet become a relevant part of the treatment landscape of unselected colorectal cancer. Beside the small subgroup of deficient mismatch repair or microsatellite instable tumors (about 5%) as a surrogate for high mutational burden and subsequently high neoantigen load and immunogenicity, inhibitors of programmed death 1 (PD-1), programmed death ligand 1 (PD-L1) and/or cytotoxic T lymphocyte-associated antigen-4 were not or only modestly effective in metastatic colorectal cancer. Thus, a variety of combination approaches with chemotherapy, targeted therapy, toll-like receptor agonists, local ablation or oncolytic viruses is currently being evaluated in different disease settings. Despite several encouraging single arm data already presented or published, available randomized data are unimpressive. Adding PD-1/PD-L1 inhibitors to fluoropyrimidines and bevacizumab maintenance showed no beneficial impact on delaying progression. In refractory disease, the combination of PD-1/PD-L1 and MEK inhibitor was not different from regorafenib, whereas a PD-1/PD-L1 and cytotoxic T lymphocyte-associated antigen-4 inhibitor combination demonstrated better overall survival compared to supportive care alone. Clinical trials in all disease settings applying different combination approaches are ongoing and may define the role of immunotherapy in colorectal cancer.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/terapia , Imunoterapia/métodos , Terapia Viral Oncolítica/métodos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/imunologia , Ensaios Clínicos como Assunto , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Terapia Combinada/métodos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Humanos , Instabilidade de Microssatélites , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/imunologia , Resultado do Tratamento
3.
Scand J Immunol ; 90(6): e12818, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31448424

RESUMO

Bladder cancer is one of the leading causes of death worldwide. The main immune mechanisms which lead to bladder cancer development or treatment outcomes have yet to be elucidated. Toll-like receptors (TLRs) play key roles against cancer. TLRs are expressed both on immune cells and on tumour cells and drive immune responses in progression as well as treatment of cancer. Identification of signalling pathways via TLRs could revolutionize further improvement of therapeutic strategies against cancers in the future. According to the recent studies, TLRs agonists are effective immunostimulants and have important role in induction of immune responses with immunotherapeutic potential against several diseases including cancer. They play an important role in the bladder urothelium as a part of immune defence against uropathogens. On the other hand, decreased TLRs expression was found in bladder tumours, particularly in non-muscle-invasive ones. Bacillus Calmette-Guerin (BCG) (agonist of TLR2 and TLR4) is approved by US FDA for immunotherapy of bladder cancer. Despite high efficiency, immunotherapy with BCG may cause toxicity and adverse effects. Nowadays, in vitro and in vivo studies have been conducted to find alternative options for non-responder patients. Studies on TLR agonists for bladder cancer treatment have shown promising results. In this review, we discuss recent data about mechanisms played by TLRs in bladder cancer developments as well as therapeutic application of TLR agonists in cancer treatment.


Assuntos
Receptores Toll-Like/metabolismo , Neoplasias da Bexiga Urinária/etiologia , Neoplasias da Bexiga Urinária/metabolismo , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais , Progressão da Doença , Suscetibilidade a Doenças , Humanos , Fenômenos Imunogenéticos , Imunoterapia/métodos , Ligantes , Terapia de Alvo Molecular , Gradação de Tumores , Estadiamento de Neoplasias , Polimorfismo Genético , Transdução de Sinais , Receptores Toll-Like/agonistas , Receptores Toll-Like/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/terapia
4.
Neuroscience ; 408: 388-399, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31026566

RESUMO

Neuroinflammation plays an important role in epileptic disorders. Toll-like receptors (TLRs) are the key signal transduction tools by which neuroinflammation may promote epileptogenesis. Depending on the stimulus nature, TLRs may engage a distinct signaling pathway. We examined the impact of early minor activation of TLR4 and TLR2 on the severity of seizure in the pilocarpine rat model of temporal lobe epilepsy (TLE). One µg of Lipopolysaccharides (LPS), Monophosphoryl lipid A (MPL), Pam3Cysor or vehicles were microinjected into the right lateral ventricle of the male Wistar rats. 24 h later, seizures were induced by intraperitoneal injection of pilocarpine, and seizure-related behaviors were monitored. 24 h after seizure induction, the hippocampal level of pro/anti-inflammatory mediators and electrophysiological properties of the dentate gyrus (DG) granular cells were investigated by western blot and whole cell patch clamp techniques, respectively. Pretreatment with TLR ligands resulted in decreased seizure severity, lower hippocampal pro-inflammatory (IL-1ß and IL-6) cytokines and higher anti-inflammatory (IL-10 and TGF- ß) mediators in the pilocarpine-treated rats. Pilocarpine induced profound hyperexcitability in the DG granule cells accompanied by potentiated excitatory postsynaptic currents (EPSCs) and dampened inhibitory postsynaptic currents (IPSCs), in contrast to the control group. However, pretreatment with TLR ligands preserved almost normal excitability and synaptic transmission against the pilocarpine. In conclusion, early activation of TLR4 and TLR2, probably through preserving normal hippocampal cytokine profile and neuronal function attenuates seizure severity in the rat model of TLE.


Assuntos
Epilepsia/fisiopatologia , Lipídeo A/análogos & derivados , Lipopolissacarídeos/farmacologia , Neurônios/efeitos dos fármacos , Convulsões/fisiopatologia , Receptores Toll-Like/agonistas , Animais , Epilepsia/induzido quimicamente , Epilepsia/tratamento farmacológico , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Lipídeo A/farmacologia , Lipídeo A/uso terapêutico , Lipopolissacarídeos/uso terapêutico , Masculino , Técnicas de Patch-Clamp , Pilocarpina , Ratos , Ratos Wistar , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico
5.
Int J Mol Sci ; 20(6)2019 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-30893757

RESUMO

In the case of epidermal barrier disruption, pathogens encounter skin-resident Langerhans cells (LCs) and are recognized by pathogen recognition receptors such as Toll-like receptors (TLRs). As the majority of microorganisms exhibit more than one TLR ligand, the mechanisms of subsequent T cell differentiation are complex and far from clear. In this study, we investigated combinatory effects on Th cell polarization by bacterial cell wall compounds peptidoglycan (PGN) and lipopolysaccharide (LPS) and by bacterial nucleic acid (DNA). Expression of maturation markers CD40, CD80, HLA-DR and CCR7 and the release of IL-1ß, IL-6 and IL-23 was strongly enhanced by simultaneous exposure to PGN, LPS and DNA in LCs. As all these factors were potential Th17 driving cytokines, we investigated the potency of combinatory TLR stimuli to induce Th17 cells via LC activation. High amounts of IL-17A and IL-22, key cytokines of Th17 cells, were detected. By intracellular costaining of IL-17⁺T cells, IL-22- (Th17) and IL-22⁺ (immature Th17) cells were identified. Interestingly, one population of LPS stimulated cells skewed into IL-9⁺Th cells, and LPS synergized with PGN while inducing high IL-22. In conclusion, our data indicates that when mediated by a fine-tuned signal integration via LCs, bacterial TLR agonists synergize and induce Th17 differentiation.


Assuntos
Células de Langerhans/citologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/imunologia , Monócitos/citologia , Peptidoglicano/farmacologia , Células Th17/imunologia , Antígenos CD/metabolismo , Ligante de CD40/metabolismo , Citocinas/metabolismo , DNA/metabolismo , Humanos , Interleucinas/metabolismo , Células de Langerhans/efeitos dos fármacos , Células de Langerhans/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Poli I-C/farmacologia , Poli U/farmacologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Regulação para Cima/efeitos dos fármacos
6.
J Vet Intern Med ; 33(2): 831-837, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30847973

RESUMO

BACKGROUND: Feline herpesvirus-1 (FHV-1) infection can result in serious morbidity and mortality, especially in kittens. Immunotherapy using liposome-toll-like receptor (TLR) ligand complexes (LTC) has been shown to activate innate immune responses. OBJECTIVES: To determine in kittens whether mucosal administration of LTC before FHV-1 inoculation would decrease severity of clinical signs and decrease quantities of FHV-1 DNA in materials collected on oropharyngeal swabs. ANIMALS: Nineteen, 14-week-old, purpose-bred kittens. METHODS: Pilot clinical trial with 2 groups of kittens allocated to either an LTC or control group. The LTC were administered into both nares and the oropharynx of the 12 LTC group kittens, and all 19 kittens were inoculated with FHV-1 24 hours later. Clinical scores were determined daily for 28 days, and oropharyngeal mucosal materials were collected every 7 days to assess FHV-1 DNA quantities for comparison between groups. RESULTS: Conjunctivitis was more common in kittens in the control group on Days 15-28 (P = .01) and Days 1-28 (P = .02). Total respiratory scores were higher in the LTC group on days 15-28 (P = .03). The LTC group had significantly decreased FHV-1 DNA on swabs when compared to the control group on some postinoculation days, using 2 methods of calculation. CONCLUSIONS AND CLINICAL IMPORTANCE: Administration of LTC to kittens was shown to decrease FHV-1 DNA and some manifestations of illness in kittens when administrated 24 hours before inoculation, suggesting clinical benefit.


Assuntos
Doenças do Gato/virologia , Infecções por Herpesviridae/veterinária , Lipossomos/administração & dosagem , Receptores Toll-Like/agonistas , Varicellovirus/imunologia , Animais , Doenças do Gato/imunologia , Doenças do Gato/prevenção & controle , Gatos , DNA Viral/isolamento & purificação , Feminino , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Imunidade Inata , Masculino , Membrana Mucosa/imunologia , Membrana Mucosa/virologia , Projetos Piloto , Varicellovirus/isolamento & purificação
7.
Mol Immunol ; 109: 27-37, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30851634

RESUMO

Small Ras GTPases are key molecules that regulate a variety of cellular responses in different cell types. Rap1 plays important functions in the regulation of macrophage biology during inflammation triggered by toll-like receptors (TLRs). However, despite sharing a relatively high degree of similarity with Rap1, no studies concerning Rap2 in macrophages and innate immunity have been reported yet. In this work, we show that either way alterations in the levels of Rap2a hampers proper macrophages response to TLR stimulation. Rap2a is activated by LPS in macrophages, and although putative activator TLR-inducible Ras guanine exchange factor RasGEF1b was sufficient to induce, it was not fully required for Rap2a activation. Silencing of Rap2a impaired LPS-induced production of IL-6 cytokine and KC/Cxcl1 chemokine, and also NF-κB activity as measured by reporter gene studies. Surprisingly, overexpression of Rap2a did also lead to marked inhibition of NF-κB activation induced by LPS, Pam3CSK4 and downstream TLR signaling molecules. We also found that Rap2a can inhibit the LPS-induced phosphorylation of the NF-κB subunit p65 at serine 536. Collectively, our data suggest that expression levels of Rap2a in macrophages might be tightly regulated to avoid unbalanced immune response. Our results implicate Rap2a in TLR-mediated responses by contributing to balanced NF-κB activity status in macrophages.


Assuntos
Regulação da Expressão Gênica , Inflamação/genética , Macrófagos/enzimologia , NF-kappa B/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Quimiocina CXCL1/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos , Macrófagos/patologia , Camundongos , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Proteínas rap de Ligação ao GTP/genética , Fatores ras de Troca de Nucleotídeo Guanina
8.
Front Immunol ; 10: 112, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30778353

RESUMO

Background and aims: Chronic hepatitis B virus (HBV) infection is a major health burden potentially evolving toward cirrhosis and hepatocellular carcinoma. HBV physiopathology is strongly related to the host immunity, yet the mechanisms of viral evasion from immune-surveillance are still misunderstood. The immune response elicited at early stages of viral infection is believed to be important for subsequent disease outcome. Dendritic cells (DCs) are crucial immune sentinels which orchestrate antiviral immunity, which offer opportunity to pathogens to subvert them to escape immunity. Despite the pivotal role of DCs in orientating antiviral responses and determining the outcome of infection, their precise involvement in HBV pathogenesis is not fully explored. Methods: One hundred thirty chronically HBV infected patients and 85 healthy donors were enrolled in the study for blood collection, together with 29 chronically HBV infected patients and 33 non-viral infected patients that were included for liver biopsy collection. In a pioneer way, we investigated the phenotypic and functional features of both circulating and intrahepatic BDCA1+ cDC2, BDCA2+ pDCs, and BDCA3+ cDC1 simultaneously in patients with chronic HBV infection by designing a unique multi-parametric flow cytometry approach. Results: We showed modulations of the frequencies and basal activation status of blood and liver DCs associated with impaired expressions of specific immune checkpoints and TLR molecules on circulating DC subsets. Furthermore, we highlighted an impaired maturation of circulating and hepatic pDCs and cDCs following stimulation with specific TLR agonists in chronic HBV patients, associated with drastic dysfunctions in the capacity of circulating DC subsets to produce IL-12p70, TNFα, IFNα, IFNλ1, and IFNλ2 while intrahepatic DCs remained fully functional. Most of these modulations correlated with HBsAg and HBV DNA levels. Conclusion: We highlight potent alterations in the distribution, phenotype and function of all DC subsets in blood together with modulations of intrahepatic DCs, revealing that HBV may hijack the immune system by subverting DCs. Our findings provide innovative insights into the immuno-pathogenesis of HBV and the mechanisms of virus escape from immune control. Such understanding is promising for developing new therapeutic strategies restoring an efficient immune control of the virus.


Assuntos
Antígenos CD1/metabolismo , Antígenos de Superfície/metabolismo , Células Dendríticas/metabolismo , Glicoproteínas/metabolismo , Vírus da Hepatite B/imunologia , Hepatite B Crônica/sangue , Hepatite B Crônica/patologia , Lectinas Tipo C/metabolismo , Fígado/patologia , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Adolescente , Adulto , Idoso , Biópsia , DNA Viral/metabolismo , Feminino , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Adulto Jovem
9.
PLoS One ; 14(2): e0208216, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794556

RESUMO

Pneumonia remains a global health threat, in part due to expanding categories of susceptible individuals and increasing prevalence of antibiotic resistant pathogens. However, therapeutic stimulation of the lungs' mucosal defenses by inhaled exposure to a synergistic combination of Toll-like receptor (TLR) agonists known as Pam2-ODN promotes mouse survival of pneumonia caused by a wide array of pathogens. This inducible resistance to pneumonia relies on intact lung epithelial TLR signaling, and inducible protection against viral pathogens has recently been shown to require increased production of epithelial reactive oxygen species (ROS) from multiple epithelial ROS generators. To determine whether similar mechanisms contribute to inducible antibacterial responses, the current work investigates the role of ROS in therapeutically-stimulated protection against Pseudomonas aerugnosa challenges. Inhaled Pam2-ODN treatment one day before infection prevented hemorrhagic lung cytotoxicity and mouse death in a manner that correlated with reduction in bacterial burden. The bacterial killing effect of Pam2-ODN was recapitulated in isolated mouse and human lung epithelial cells, and the protection correlated with inducible epithelial generation of ROS. Scavenging or targeted blockade of ROS production from either dual oxidase or mitochondrial sources resulted in near complete loss of Pam2-ODN-induced bacterial killing, whereas deficiency of induced antimicrobial peptides had little effect. These findings support a central role for multisource epithelial ROS in inducible resistance against a bacterial pathogen and provide mechanistic insights into means to protect vulnerable patients against lethal infections.


Assuntos
Imunidade nas Mucosas/efeitos dos fármacos , Lipopeptídeos/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Pneumonia Bacteriana/imunologia , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/imunologia , Receptores Toll-Like/agonistas , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Infecções Bacterianas/patologia , Células Cultivadas , Citoproteção/efeitos dos fármacos , Citoproteção/imunologia , Combinação de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células HEK293 , Humanos , Imunidade nas Mucosas/fisiologia , Exposição por Inalação , Ligantes , Lipopeptídeos/administração & dosagem , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/administração & dosagem , Pneumonia Bacteriana/metabolismo , Pneumonia Bacteriana/patologia , Substâncias Protetoras/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Vacinação/métodos
10.
J Vet Intern Med ; 33(2): 838-845, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30770582

RESUMO

BACKGROUND: Nonspecific induction of local innate immune responses by mucosally administered immunotherapy is a new approach to protection from upper respiratory tract infections. Therefore, a new liposome-toll-like receptor complex (LTC) immune stimulant was developed and investigated for its ability to activate innate immune responses in cats, both in vitro and in vivo, as part of an initial evaluation of LTC for use as an immunotherapeutic agent in cats. OBJECTIVES: We hypothesized that LTC could activate innate immune responses in cats after topical application to nasal and oropharyngeal mucosal surfaces. ANIMALS: Mucosal immune responses to topical administration of LTC were assessed in 7 healthy, purpose-bred cats, and in vitro responses were assessed using blood samples from healthy cats. METHODS: Cytokine and cellular immune responses to LTC were evaluated in blood samples, nasal lavage specimens, and pharyngeal swabs from cats, using reverse transcriptase polymerase chain reaction assays, ELISA assays, and flow cytometry. RESULTS: Liposome-TLR complexes rapidly activated leukocytes in vitro, including upregulation of costimulatory molecule expression and cytokine production. Topical administration of LTC in healthy cats triggered rapid recruitment of monocytes to the nasal and oropharyngeal mucosa. CONCLUSIONS AND CLINICAL IMPORTANCE: Liposome-TLR complexes were found to effectively activate innate immune responses in cats after mucosal administration. These findings suggest that LTC have potential for use as a new mucosally administered immunotherapy for nonspecific protection from viral and bacterial respiratory tract infections.


Assuntos
Gatos/imunologia , Imunidade Inata/efeitos dos fármacos , Lipossomos/administração & dosagem , Mucosa Respiratória/efeitos dos fármacos , Receptores Toll-Like/agonistas , Administração através da Mucosa , Animais , Citocinas/análise , Ensaio de Imunoadsorção Enzimática/veterinária , Citometria de Fluxo/veterinária , Leucócitos/imunologia , Ligantes , Mucosa Respiratória/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária
11.
Blood Adv ; 3(2): 122-131, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30647074

RESUMO

Patients with myeloproliferative neoplasms (MPN) have high levels of inflammatory cytokines, some of which drive many of the debilitating constitutional symptoms associated with the disease and may also promote expansion of the neoplastic clone. We report here that monocytes from patients with MPN have defective negative regulation of Toll-like receptor (TLR) signaling that leads to unrestrained production of the inflammatory cytokine tumor necrosis factor α (TNF-α) after TLR activation. Specifically, monocytes of patients with MPN are insensitive to the anti-inflammatory cytokine interleukin 10 (IL-10) that negatively regulates TLR-induced TNF-α production. This inability to respond to IL-10 is a not a direct consequence of JAK2 V617F , as the phenotype of persistent TNF-α production is a feature of JAK2 V617F and wild-type monocytes alike from JAK2 V617F -positive patients. Moreover, persistent TNF-α production was also discovered in the unaffected identical twin of a patient with MPN, suggesting it could be an intrinsic feature of those predisposed to acquire MPN. This work implicates sustained TLR signaling as not only a contributor to the chronic inflammatory state of MPN patients but also a potential predisposition to acquire MPN.


Assuntos
Transtornos Mieloproliferativos/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Alelos , Animais , Citocinas/metabolismo , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Macrófagos/metabolismo , Camundongos , Monócitos/metabolismo , Transtornos Mieloproliferativos/etiologia , Ligação Proteica , Receptores de Interleucina-10/metabolismo , Receptores Toll-Like/agonistas
12.
Nutrients ; 11(2)2019 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-30678156

RESUMO

BACKGROUND: Pycnogenol® (PYC), an extract of French maritime pine bark, is widely used as a dietary supplement. PYC has been shown to exert anti-inflammatory actions via inhibiting the Toll-like receptor 4 (TLR4) pathway. However, the role of the other receptors from the TLR family in the immunomodulatory activity of PYC has not been described so far. AIM: The aim of this study was to investigate whether PYC might exert its immunomodulatory properties through cell membrane TLRs (TLR1/2, TLR5, and TLR2/6) other than TLR4. Moreover, the effect of gastrointestinal metabolism on the immunomodulatory effects of PYC was investigated. FINDINGS: We showed that intact non-metabolized PYC dose-dependently acts as an agonist of TLR1/2 and TLR2/6 and as a partial agonist of TLR5. PYC on its own does not agonize or antagonize TLR4. However, after the formation of complexes with lipopolysaccharides (LPS), it is a potent activator of TLR4 signaling. Gastrointestinal metabolism of PYC revealed the immunosuppressive potential of the retentate fraction against TLR1/2 and TLR2/6 when compared to the control fraction containing microbiota and enzymes only. The dialyzed fraction containing PYC metabolites revealed the capacity to induce anti-inflammatory IL-10 secretion. Finally, microbially metabolized PYC affected the colonic microbiota composition during in vitro gastrointestinal digestion. CONCLUSIONS: This study showed that gastrointestinal metabolism of PYC reveals its biological activity as a potential inhibitor of TLRs signaling. The results suggest that metabolized PYC acts as a partial agonist of TLR1/2 and TLR2/6 in the presence of the microbiota-derived TLR agonists (retentate fraction) and that it possesses anti-inflammatory potential reflected by the induction of IL-10 from THP-1 macrophages (dialysate fraction).


Assuntos
Flavonoides/farmacologia , Extratos Vegetais/farmacologia , Receptores Toll-Like/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Flavonoides/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Imunomodulação , Lipopolissacarídeos/química , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Extratos Vegetais/administração & dosagem , Receptores Toll-Like/agonistas
13.
Cancer Res ; 79(1): 159-170, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30224373

RESUMO

Metastatic cancer involving spread to the peritoneal cavity is referred to as peritoneal carcinomatosis and has a very poor prognosis. Activating the antitumor immune response in the characteristically immune-suppressive peritoneal environment presents a potential strategy to treat this disease. In this study, we show that a toll-like receptor (TLR) and C-type lectin receptor (CLR) agonist pairing of monophosphoryl lipid A (MPL) and trehalose-6,6'-dicorynomycolate (TDCM) effectively inhibits tumor growth and ascites development in a mouse model of aggressive mammary cancer-induced peritoneal carcinomatosis. MPL/TDCM treatment similarly inhibited peritoneal EL4 tumor growth and ascites development. These effects were not observed in mice lacking B cells or mice lacking CD19, which are deficient in B-1a cells, an innate-like B-cell population enriched in the peritoneal cavity. Remarkably, adoptive transfer of B-1a cells, but not splenic B cells from WT mice, restored MPL/TDCM-induced protection in mice with B-cell defects. Treatment induced B-1 cells to rapidly produce high levels of natural IgM reactive against tumor-associated carbohydrate antigens. Consistent with this, we found significant deposition of IgM and C3 on peritoneal tumor cells as early as 5 days post-treatment. Mice unable to secrete IgM or complement component C4 were not protected by MPL/TDCM treatment, indicating tumor killing was mediated by activation of the classical complement pathway. Collectively, our findings reveal an unsuspected role for B-1 cell-produced natural IgM in providing protection against tumor growth in the peritoneal cavity, thereby highlighting potential opportunities to develop novel therapeutic strategies for the prevention and treatment of peritoneal metastases. SIGNIFICANCE: This work identifies a critical antitumor role for innate-like B cells localized within the peritoneal cavity and demonstrates a novel strategy to activate their tumor-killing potential.See related commentary by Tripodo, p. 5.


Assuntos
Subpopulações de Linfócitos B/imunologia , Imunidade Inata/imunologia , Imunoglobulina M/imunologia , Ativação Linfocitária/imunologia , Neoplasias Mamárias Animais/imunologia , Cavidade Peritoneal/patologia , Neoplasias Peritoneais/imunologia , Animais , Subpopulações de Linfócitos B/efeitos dos fármacos , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/patologia , Fatores Corda/farmacologia , Feminino , Imunidade Inata/efeitos dos fármacos , Imunoglobulina M/efeitos dos fármacos , Lectinas Tipo C/agonistas , Lipídeo A/análogos & derivados , Lipídeo A/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Receptores Toll-Like/agonistas
14.
J Immunol Methods ; 464: 131-137, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30395817

RESUMO

Cytokine production by human peripheral blood mononuclear cells including monocytes, is frequently assessed by measuring secreted cytokines using enzyme linked immunosorbent assay (ELISA), whereby the total concentration of one cytokine of interest is obtained without information regarding the cell type responsible for making the cytokine. Cytokines can be retained inside the cell using protein transport inhibitors. Subsequent analysis by flow cytometry not only identifies the cell type producing the cytokine but can semi-quantitate the amount of cytokine produced by measuring the geometric mean fluorescence intensity (gMFI) and is amenable to analyzing more than one protein associated with the same cell (multiplexing). We hypothesized that a more comprehensive and biologically meaningful cytokine profile could be acquired by measuring both secreted and the retained intracellular cytokines in parallel cultures of magnetic-sorted CD14+ monocytes. Peripheral monocytes were isolated from 18 healthy donors and treated with standardized molecules that stimulate cytokine production; Toll-like receptor (TLR)4 agonist (lipopolysaccharide, LPS) or TLR7/8 agonist (R848). Pro-inflammatory cytokines (interleukin (IL)-6, IL-8 and tumour necrosis factor (TNF)) secreted into the culture medium were measured by ELISA. Parallel cultures were treated with LPS and R848 in the presence of brefeldin A (protein transport inhibitor) and the accumulated intracellular cytokines measured by flow cytometry. Each cytokine (IL-6/IL-8/TNF) gave a unique general pattern when secreted versus intracellular cytokine measurements (frequency and gMFI) were plotted to determine correlation. For monocytes treated with the TLR4 agonist, secreted IL-8 correlated with the frequency of IL-8 positive cells (R = 0.559, p = .016) and not with the amount (gMFI) of IL-8 per cell. In contrast, monocytes treated with the TLR7/8 agonist showed no correlation of secreted IL-8 with the frequency of IL-8 positive cells, but with this treatment secreted IL-6 was correlated with an increase in the frequency of IL-6 positive cells (R = 0.501, p = .034). TNF secretion from monocytes treated with either the TLR4 or TLR7/8 agonist did not correlate with the frequency or gMFI of TNF positive cells. However, there were significant correlations between the TLR4 and TLR7/8 induced TNF response (secreted and gMFI). We conclude that there are fundamental differences in secreted and intracellular IL-6/IL-8/TNF production after monocytes are treated with TLR agonists. Furthermore, secreted and intracellular cytokine analyses are complementary measures that should be used in parallel to explore inflammatory response and cytokine biology.


Assuntos
Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imidazóis/farmacologia , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Receptores Toll-Like/agonistas , Adulto , Células Cultivadas , Citocinas/imunologia , Humanos , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Via Secretória , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Adulto Jovem
15.
Mol Immunol ; 106: 87-98, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30593933

RESUMO

TLR21 can recognize unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODN) and activates NF-κB immune signaling pathway. However, the function of TLR21 in duck remains largely unclear. Here, the complete duck TLR21 (duTLR21) cDNA was cloned from Cherry Valley duck for the first time, and its immune response was preliminarily studied. Tissue specificity analysis showed duTLR21 was higher expressed in the peripheral blood, spleen, bursa of Fabricius and cecum. The expression of duTLR21 was significantly upregulated after stimulation with CpG-ODN or duck plague virus (DPV), but not Tembusu virus (TMUV), LPS or Poly (I:C). In addition, the transfection of DEF with duTLR21 stimulated by CpG-ODN activated NF-κB, through this signal pathway, the transcription of IL-1ß, IL-6 and IFN-α were promoted, whereas knockdown of duTLR21 impaired the transcription of these genes. Furthermore, the overexpression of duTLR21 inhibited the replication of the DPV and the knockdown of duTLR21 by shRNA significantly promoted DPV replication in vitro. Altogether, these results indicate that duTLR21 can be activated by CpG-ODN, which mediates activation of NF-κB signaling pathway, and plays an important role in the host defence of DPV infection.


Assuntos
Alphaherpesvirinae/fisiologia , Proteínas Aviárias/imunologia , Patos/imunologia , Infecções por Herpesviridae/imunologia , NF-kappa B/imunologia , Oligodesoxirribonucleotídeos/farmacocinética , Doenças das Aves Domésticas/imunologia , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/imunologia , Replicação Viral/efeitos dos fármacos , Animais , Proteínas Aviárias/agonistas , Proteínas Aviárias/genética , Citocinas/genética , Citocinas/imunologia , Patos/genética , Patos/virologia , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/veterinária , NF-kappa B/genética , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/patologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/genética , Replicação Viral/genética , Replicação Viral/imunologia
16.
Molecules ; 23(12)2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30518153

RESUMO

A series of nine derivatives (2⁻10) were prepared from the diterpene solidagenone (1) and their structures were elucidated by means of spectroscopic studies. Their ability to inhibit inflammatory responses elicited in peritoneal macrophages by TLR ligands was investigated. Compounds 5 and 6 showed significant anti-inflammatory effects, as they inhibited the protein expression of nitric oxide synthase (NOS-2), cyclooxygenase-2 (COX-2), and cytokine production (TNF-α, IL-6, and IL-12) induced by the ligand of TLR4, lipopolysaccharide (LPS), acting at the transcriptional level. Some structure⁻activity relationships were outlined. Compound 5 was selected as a representative compound and molecular mechanisms involved in its biological activity were investigated. Inhibition of NF-κB and p38 signaling seems to be involved in the mechanism of action of compound 5. In addition, this compound also inhibited inflammatory responses mediated by ligands of TLR2 and TLR3 receptors. To rationalize the obtained results, molecular docking and molecular dynamic studies were carried out on TLR4. All these data indicate that solidagenone derivative 5 might be used for the design of new anti-inflammatory agents.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Furanos/química , Furanos/farmacologia , Naftalenos/química , Naftalenos/farmacologia , Receptores Toll-Like , Animais , Células Cultivadas , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Receptores Toll-Like/agonistas , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/metabolismo
17.
Biomed Res Int ; 2018: 5693736, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30539014

RESUMO

Activation of toll-like receptors (TLRs) has been shown to play an important role in leishmaniosis by enhancing the parasite specific immune responses to control infection. However, the role of TLR agonists has not been studied in detail in dogs. The aim of this study was to determine the effect of TLR3, TLR4, and TLR7 agonists (TLR3a, TLR4a, and TLR7a) alone or in combination with Leishmania infantum antigen (LSA) on TNF-α and IL-6 production in blood from dogs living in endemic areas of canine leishmaniosis (CanL). Twenty-four healthy dogs from Catalonia (n=14) and Ibizan hound dogs from the island of Mallorca (n=10) were enrolled. Whole blood with TLR3a, TLR4a, and TLR7a alone or combined with LSA were cultured separately, and IFN-γ, TNF-α, and IL-6 were measured by ELISA. A significant increase of TNF-α was found for all conditions studied compared to medium alone. Stimulation with TLR4a (p=0.0001) and TLR7a (p=0.005) presented a significantly marked increase in TNF-α and IL-6 production compared to TLR3a. Importantly, significantly higher TNF-α production was found in LSA+TLR4a (p=0.0001) stimulated blood and LSA+TLR7a (p=0.005) compared to LSA alone. All dogs showed higher TNF-α production after LSA+TLR7a compared to TLR7a (p=0.047) and LSA+TLR3a compared to TLR3a (p=0.052). These data indicate a marked inflammatory cytokine effect of TLR4a and TLR7a on blood from healthy dogs living in endemic areas of CanL. Additionally, LSA+TLR7a promoted a synergistic proinflammatory effect with TNF-α in all dogs. Those findings suggest an active role of TLRs in proinflammatory responses, which might be strongly involved in the process of disease resolution.


Assuntos
Antígenos de Protozoários/sangue , Antígenos de Protozoários/imunologia , Citocinas/sangue , Leishmania infantum/imunologia , Receptores Toll-Like/agonistas , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Doenças do Cão/sangue , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Cães , Feminino , Interferon gama/biossíntese , Interleucina-6/sangue , Leishmaniose/veterinária , Masculino , Parasitos/imunologia , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/sangue
18.
J Immunol ; 201(12): 3741-3749, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30397036

RESUMO

Therapy with tumor-specific Abs is common in the clinic but has limited success against solid malignancies. We aimed at improving the efficacy of this therapy by combining a tumor-specific Ab with immune-activating compounds. In this study, we demonstrate in the aggressive B16F10 mouse melanoma model that concomitant application of the anti-TRP1 Ab (clone TA99) with TLR3-7/8 or -9 ligands, and IL-2 strongly enhanced tumor control in a therapeutic setting. Depletion of NK cells, macrophages, or CD8+ T cells all mitigated the therapeutic response, showing a coordinated immune rejection by innate and adaptive immune cells. FcγRs were essential for the therapeutic effect, with a dominant role for FcγRI and a minor role for FcγRIII and FcγRIV. FcγR expression on NK cells and granulocytes was dispensable, indicating that other tumoricidal functions of NK cells were involved and implicating that FcγRI, -III, and -IV exerted their activity on macrophages. Indeed, F4/80+Ly-6C+ inflammatory macrophages in the tumor microenvironment displayed high levels of these receptors. Whereas administration of the anti-TRP1 Ab alone reduced the frequency of these macrophages, the combination with a TLR agonist retained these cells in the tumor microenvironment. Thus, the addition of innate stimulatory compounds, such as TLR ligands, to tumor-specific Ab therapy could greatly enhance its efficacy in solid cancers via optimal exploitation of FcγRs.


Assuntos
Anticorpos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Imunoterapia/métodos , Interleucina-2/uso terapêutico , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Melanoma/terapia , Receptores de IgG/metabolismo , Animais , Antígenos de Neoplasias/imunologia , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunização , Masculino , Melanoma/imunologia , Melanoma Experimental , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases/imunologia , Receptores de IgG/genética , Receptores Toll-Like/agonistas
19.
Cancer Treat Rev ; 71: 88-101, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30390423

RESUMO

The field of cancer immunotherapy has been revolutionized with the use of immune checkpoint blockade antibodies such as anti-programmed cell death 1 protein (PD-1) and chimeric antigen receptor T cells. Significant clinical benefits are observed in different cancer types with these treatments. While considerable efforts are made in augmenting tumor-specific T cell responses with these therapies, other immunotherapies that actively stimulate endogenous anti-tumor T cells and generating long-term memory have received less attention. Given the high cost of cancer immunotherapies especially with chimeric antigen receptor T cells, not many patients will have access to such treatments. The next-generation of cancer immunotherapy could entail in vivo cancer vaccination to activate both the innate and adaptive anti-tumor responses. This could potentially be achieved via in vivo targeting of dendritic cells which are an indispensable link between the innate and adaptive immunities. Dendritic cells highly expressed toll-like receptors for recognizing and eliminating pathogens. Synthetic toll-like receptors agonists could be synthesized at a low cost and have shown promise in preclinical and clinical trials. As different subsets of human dendritic cells exist in the immune system, activation with different toll-like receptor agonists could exert profound effects on the quality and magnitude of anti-tumor T cell responses. Here, we reviewed the different subsets of human dendritic cells. Using published preclinical and clinical cancers studies available on PubMed, we discussed the use of clinically approved and emerging toll-like receptor agonists to activate dendritic cells in vivo for cancer immunotherapy. Finally, we searched www.clinicaltrials.gov and summarized the active cancer trials evaluating toll-like receptor agonists as an adjuvant.


Assuntos
Células Dendríticas/imunologia , Imunoterapia/métodos , Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Receptores Toll-Like/agonistas , Animais , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Vacinas Anticâncer , Humanos , Imiquimode/farmacologia , Células de Langerhans/citologia , Células de Langerhans/efeitos dos fármacos , Células de Langerhans/imunologia , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Pele/citologia , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/imunologia , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/agonistas , Receptor 8 Toll-Like/imunologia , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/imunologia
20.
Expert Opin Ther Pat ; 28(11): 837-847, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30280939

RESUMO

INTRODUCTION: Currently, there is no efficient vaccine available against clinical malaria. However, continuous efforts have been committed to develop powerful antimalarial vaccine by discovery of novel antigens with in-depth understanding of its nature, immunogenicity, and presentation (delivery adjuvants). Moreover, another important part of vaccine development includes discovery of better immunostimulatory formulation components (immunostimulants). A protective vaccine against malaria requires antigen-specific B and T helper cell responses as well as cytotoxic T lymphocyte (CTL) responses. A long-lasting B and T memory cell production is also required for effective malaria vaccine. Since activation of Toll-like receptors (TLRs) promotes both innate inflammatory responses as well as the induction of adaptive immunity, several initiatives have been mounted during the last few years for the use of TLR agonists as malaria vaccine adjuvants. AREAS COVERED: The review summarizes reports related to the use and development of TLR agonists as malaria vaccine adjuvants and describes various strategies involved for the selection of specific antigens and TLR agonists. EXPERT OPINION: TLR agonists are promising adjuvants for the development of effective malaria vaccine, allowing for both innate inflammatory responses as well as the induction of adaptive immunity.


Assuntos
Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Receptores Toll-Like/agonistas , Imunidade Adaptativa/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos/imunologia , Desenho de Drogas , Humanos , Imunidade Inata/imunologia , Malária/imunologia , Patentes como Assunto
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