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1.
Chem Commun (Camb) ; 58(24): 3925-3928, 2022 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-35244125

RESUMO

Adjuvants are important components in vaccines to increase the immunogenicity of proteins and induce optimal immunity. In this study, we designed a novel ternary adjuvant system Alum + c-GAMP + poly(I:C) with STING agonist 3,3'-c-GAMP (c-GAMP) and TLR3 agonist poly(I:C) co-adsorbed on the conventional adjuvant aluminum gel (Alum), and further constructed an S1 protein vaccine. Two doses of vaccination with the ternary adjuvant vaccine were sufficient to induce a balanced Th1/Th2 immune response and robust humoral and cellular immunity. Additionally, the ternary adjuvant group had effective neutralizing activity against live virus SARS-CoV-2 and pseudovirus of all variants of concern (alpha, beta, gamma, delta and omicron). These results indicate that the ternary adjuvants have a significant synergistic effect and can rapidly trigger potent immune responses; the combination of the ternary adjuvant system with S1 protein is a promising COVID-19 vaccine candidate.


Assuntos
COVID-19 , SARS-CoV-2 , Adjuvantes Imunológicos/farmacologia , Compostos de Alúmen , Alumínio , Animais , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19/farmacologia , Humanos , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Poli I
2.
J Feline Med Surg ; 24(6): e43-e56, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35302413

RESUMO

OBJECTIVES: Feline autologous mesenchymal stem cells (MSCs) show promise for immunomodulatory activity, but the functional impact of chronic kidney disease (CKD), concurrent immunosuppressive drug administration or infection is unknown. The study objectives compare endogenous cytokine gene expression (interleukin [IL]-6, IL-10, IL-12p40, IL-18 and transforming growth factor beta [TGF-ß]) in adipose-derived MSCs (aMSCs) from cats with and without CKD, following in vitro exposure to microbial ligands and treatment with common immunosuppressive drugs. METHODS: Previously obtained aMSCs, phenotype CD44+, CD90+, CD105+ and MHCII-, from cats with (n = 6) and without (n = 6) CKD were compared via real-time PCR (RT-PCR) for immunomodulatory gene expression. aMSCs were exposed in vitro to lipopolysaccharide (LPS), peptidoglycan or polyinosinic:polycytidylic acid (Poly I:C), simulating bacterial or viral exposure, respectively. aMSCs were also exposed to ciclosporin, dexamethasone or methotrexate. Gene expression was measured using RT-PCR, and Cq was utilized after each run to calculate the delta cycle threshold. RESULTS: aMSCs isolated from healthy and CKD cats showed no significant differences in gene expression in the five measured cytokines. No significant changes in measured gene expression after drug treatment or microbial ligand stimulation were observed between normal or CKD affected cats. Proinflammatory genes (IL-6, IL-12p40 and IL-18) showed altered expression in aMSCs from both groups when compared with the same cells in standard culture after exposure to methotrexate. Poly I:C altered IL-6 and TGF-ß gene expression in aMSCs from both healthy and CKD cats when compared with the same cells in standard culture. CONCLUSIONS AND RELEVANCE: The five genes tested showed no statistical differences between aMSCs from healthy or CKD cats. There was altered cytokine gene expression between the control and treatment groups of both healthy and CKD cats suggesting feline aMSCs have altered function with immunosuppressive treatment or microbial ligand exposure. Although the current clinical relevance of this pilot study comparing brief exposure to select agents in vitro in aMSCs from a small number of cats is unknown, the study highlights a need for continued investigation into the effects of disease and concurrent therapies on use of cell-based therapies in feline patients.


Assuntos
Doenças do Gato , Células-Tronco Mesenquimais , Insuficiência Renal Crônica , Tecido Adiposo , Animais , Doenças do Gato/tratamento farmacológico , Doenças do Gato/genética , Gatos , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-18/metabolismo , Interleucina-6/metabolismo , Ligantes , Metotrexato/metabolismo , Preparações Farmacêuticas/metabolismo , Projetos Piloto , Poli I/metabolismo , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/terapia , Insuficiência Renal Crônica/veterinária , Fator de Crescimento Transformador beta
3.
Biol Open ; 11(3)2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35107124

RESUMO

Protein malnourishment and immune stress are potent perinatal stressors, encountered by children born under poor socioeconomic conditions. Thus, it is necessary to investigate how such stressors synergistically contribute towards developing neurological disorders in affected individuals. Pups from Wistar females, maintained on normal (high-protein, HP:20%) and low-protein (LP:8%) diets were used. Single and combined exposures of Poly I:C (viral mimetic: 5 mg/kg body weight) and Lipopolysaccharide (LPS; bacterial endotoxin: 0.3 mg/kg body weight) were injected to both HP and LP pups at postnatal days (PND) 3 and 9 respectively, creating eight groups: HP (control); HP+Poly I:C; HP+LPS; HP+Poly I:C+LPS; LP; LP+Poly I:C; LP+LPS; LP+Poly I:C+LPS (multi-hit). The effects of stressors on hippocampal cytoarchitecture and behavioral abilities were studied at PND 180. LP animals were found to be more vulnerable to immune stressors than HP animals and symptoms like neuronal damage, spine loss, downregulation of Egr 1 and Arc proteins, gliosis and behavioral deficits were maximum in the multi-hit group. Thus, from these findings it is outlined that cellular and behavioral changes that occur following multi-hit exposure may predispose individuals to developing Schizophrenia-like pathologies during adulthood.


Assuntos
Esquizofrenia , Animais , Peso Corporal , Feminino , Lipopolissacarídeos/efeitos adversos , Neurônios , Poli I , Gravidez , Esquizofrenia/diagnóstico , Esquizofrenia/etiologia
4.
J Gerontol A Biol Sci Med Sci ; 77(2): 276-282, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-34626114

RESUMO

Metformin, a clinical agent of type 2 diabetes, is reported as a potential geroprotector. Viral infection induces phenotypes of senescence in human T cells, and polyinosinic:polycytidylic acid (poly I:C), a viral mimic, induces upregulation of senescence-associated beta-galactosidase (SA-ß-gal) activity in the ovary of the annual fish Nothobranchius guentheri. However, the effects and mechanisms of metformin on poly I:C-induced aging-like phenomena are poorly understood in vertebrates. In this study, the activity of SA-ß-gal increased in the gut of 12-month-old fish and poly I:C-injected 6-month-old fish, compared to 6-month-old control fish, indicating that poly I:C induces aging-like phenomena in the gut of the fish. Metformin supplementation retarded accumulation of SA-ß-gal in the gut of old fish and poly I:C-treated young fish. The results of qPCR analysis showed that metformin reduced NF-κB-mediated inflammatory response including the decreased level of proinflammatory cytokine IL-8 and increased expression of anti-inflammatory cytokine IL-10 in the gut of the fish with natural aging and poly I:C-injected 6-month-old fish. Metformin also exhibited antioxidant effects, as it reduced reactive oxygen species production that is associated with the upregulation of FoxO3a and PGC-1α in the gut of 6-month-old fish with poly I:C injection. Expression of AMPK and SIRT1 was reduced in the gut of 6-month-old fish with poly I:C treatment, and feeding metformin reversed these declines. Taken together, the present study suggested that poly I:C injection led to aging-like phenomena in the gut and metformin activated AMPK and SIRT1 to reduce NF-κB-mediated inflammation and resist oxidative stress via enhanced expression of FoxO3a and PGC-1α and finally delayed gut aging in vertebrates.


Assuntos
Diabetes Mellitus Tipo 2 , Metformina , Proteínas Quinases Ativadas por AMP/metabolismo , Envelhecimento , Animais , Citocinas/metabolismo , Feminino , Inflamação , Metformina/farmacologia , NF-kappa B/metabolismo , Estresse Oxidativo , Poli I/metabolismo , Poli I/farmacologia , Sirtuína 1/metabolismo
5.
Mol Cells ; 45(4): 257-272, 2022 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-34949739

RESUMO

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNß, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.


Assuntos
Morte Celular Autofágica , Receptor 3 Toll-Like , Inibidores de Caspase/metabolismo , Inibidores de Caspase/farmacologia , Caspases/metabolismo , Ligantes , Lipopolissacarídeos/farmacologia , Macrófagos , Poli I/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor 3 Toll-Like/metabolismo
6.
Cells ; 10(3)2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33801464

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.


Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteína DEAD-box 58/metabolismo , Interferons/metabolismo , Receptores Imunológicos/metabolismo , SARS-CoV-2/metabolismo , Proteína DEAD-box 58/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Interferons/genética , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Fosfoproteínas/metabolismo , Poli C/farmacologia , Poli I/farmacologia , Regiões Promotoras Genéticas , /metabolismo , Receptores Imunológicos/genética , SARS-CoV-2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima , Zika virus/genética , Zika virus/metabolismo
7.
Microb Pathog ; 150: 104716, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33383149

RESUMO

The IL-33/ST2 axis is known to be involved in liver pathologies and IL-33 is over-expressed in mouse hepatitis models. We aimed to investigate the proposed protective effect of IL-33 in murine fulminant hepatitis induced by a Toll like receptor 3 (TLR3) viral mimetic, Poly I:C or by Concanavalin-A (ConA). The Balb/C mice were administered intravenously with ConA (15 mg/kg) or Poly I:C (30 µg/mouse) to induce acute hepatitis along with vehicle control. The recombinant mouse IL-33 (rIL-33) was injected (0.2 µg/mouse) to mice 2 h prior to ConA or Poly I:C injection to check its hepato-protective effects. The gross lesions, level of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), histopathology (H&E staining) and levels of IFNγ and TNFα were measured by ELISA. The gross pathological liver injury induced by Poly I:C or ConA was reduced by rIL-33 administration in mice. The levels of AST and ALT were significantly (P ≤ 0.05) higher in mice challenged with Poly I:C or ConA in comparison to control mice. The rIL-33 pre-treated mice in both Poly I:C and ConA challenge groups showed significantly (P ≤ 0.05) lower levels of AST and ALT, and decreased liver injury (parenchymal and per-vascular necrotic areas) in histological liver sections. The soluble levels of TNFα and IFNγ were significantly (P ≤ 0.05) raised in Poly I:C or ConA challenged mice than control mice. The levels of TNFα and IFNγ were significantly reduced (P ≤ 0.05) in rIL-33 pre-treated mice. In conclusion, the exogenous IL-33 administration mitigated liver injury and inflammation (decreased levels of IFNγ and TNFα) in Poly I:C and ConA-induced acute hepatitis in mice.


Assuntos
Hepatite , Interleucina-33 , Animais , Concanavalina A/toxicidade , Hepatite/prevenção & controle , Inflamação/tratamento farmacológico , Fígado , Camundongos , Poli I
8.
Sci Transl Med ; 12(565)2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33055241

RESUMO

Intratumoral therapies, especially Toll-like receptor agonists, can trigger both the innate and adaptive immune systems. BO-112 is a nanoplexed form of polyinosinic:polycytidylic acid (poly I:C) that induces local and systemic immunotherapeutic effects in mouse models. In a multicenter phase 1 clinical trial, repeated intratumoral administrations of BO-112 induced an increase in tumor cell necrosis and apoptosis, as well as augmented immune reactivity according to gene expression profiling. The first three cohorts receiving BO-112 as a monotherapy resulted in a recommended dose of 1 mg that could be safely repeated. Two grade 3 to 4 adverse reactions in the form of reversible thrombocytopenia were reported. In a fourth cohort of 28 patients with tumors that had primary resistance to anti-programmed cell death protein-1 (PD-1), the combination of intratumoral BO-112 with nivolumab or pembrolizumab was also well tolerated, and 3 patients (2 with melanoma and 1 with renal cell carcinoma) achieved partial responses, with 10 more patients having stable disease at 8 to 12 weeks. Thus, local BO-112 combined with a systemic anti-PD-1 agent might be a strategy to revert anti-PD-1 resistance.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Melanoma , Animais , Humanos , Melanoma/tratamento farmacológico , Camundongos , Nivolumabe/uso terapêutico , Poli I
9.
Mol Pharm ; 17(10): 3794-3812, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-32841040

RESUMO

We have developed a macromolecular prodrug platform based on poly(l-lysine succinylated) (PLS) that targets scavenger receptor A1 (SR-A1), a receptor expressed by myeloid and endothelial cells. We demonstrate the selective uptake of PLS by murine macrophage, RAW 264.7 cells, which was eliminated upon cotreatment with the SR-A inhibitor polyinosinic acid (poly I). Further, we observed no uptake of PLS in an SR-A1-deficient RAW 264.7 cell line, even after 24 h incubation. In mice, PLS distributed to lymphatic organs following i.v. injection, as observed by ex vivo fluorescent imaging, and accumulated in lymph nodes following both i.v. and i.d. administrations, based on immunohistochemical analysis with high-resolution microscopy. As a proof-of-concept, the HIV antiviral emtricitabine (FTC) was conjugated to the polymer's succinyl groups via ester bonds, with a drug loading of 14.2% (wt/wt). The prodrug (PLS-FTC) demonstrated controlled release properties in vitro with a release half-life of 15 h in human plasma and 29 h in esterase-inhibited plasma, indicating that drug release occurs through both enzymatic and nonenzymatic mechanisms. Upon incubation of PLS-FTC with human peripheral blood mononuclear cells (PBMCs), the released drug was converted to the active metabolite FTC triphosphate. In a pharmacokinetic study in rats, the prodrug achieved ∼7-19-fold higher concentrations in lymphatic tissues compared to those in FTC control, supporting lymphatic-targeted drug delivery. We believe that the SR-A1-targeted macromolecular PLS prodrug platform has extraordinary potential for the treatment of infectious diseases.


Assuntos
Fármacos Anti-HIV/administração & dosagem , Portadores de Fármacos/química , Infecções por HIV/tratamento farmacológico , Receptores Depuradores Classe A/metabolismo , Animais , Fármacos Anti-HIV/farmacocinética , Liberação Controlada de Fármacos , Emtricitabina/administração & dosagem , Emtricitabina/farmacocinética , Feminino , Meia-Vida , Humanos , Masculino , Camundongos , Poli I/farmacologia , Polilisina/química , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacocinética , Estudo de Prova de Conceito , Células RAW 264.7 , Ratos , Receptores Depuradores Classe A/antagonistas & inibidores , Receptores Depuradores Classe A/genética
10.
Biosci Biotechnol Biochem ; 82(11): 1889-1901, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30079840

RESUMO

Polyinosinic-polycytidylic acid (PIC), a double-stranded RNA that induces innate immunity in mammals, is a candidate immunopotentiator for pharmaceuticals. The potency and adverse effects of PIC are strongly correlated with the nucleotide length, and the inability to precisely control the length in PIC production limits its practical use. Length extension during the annealing process is the major factor underlying the lack of control, but tuning the annealing conditions is insufficient to resolve this issue. In this study, we developed a novel method to produce accurate nucleotide length PIC at an industrial scale. The length extension was significantly suppressed by the assembly of multiple short polyinosinic acid molecules with one long polycytidylic acid molecule. A newly developed PIC, uPIC100-400, demonstrated a reproducible length and better storage stability than that of corresponding evenly structured PIC. Human dsRNA receptors exhibited equivalent responsiveness to uPIC100-400 and the evenly structured PIC with the same length.


Assuntos
Conformação de Ácido Nucleico , Nucleotídeos/química , Poli I-C/química , Poli I-C/síntese química , Linhagem Celular , Temperatura Alta , Humanos , Imunidade Inata , Poli C/química , Poli I/química , RNA de Cadeia Dupla/química
11.
J Immunol ; 199(4): 1261-1274, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28696256

RESUMO

Dead cells accumulating in the tissues may contribute to chronic inflammation. We examined the cause of impaired apoptotic cell clearance in human and murine lupus. Dead cells accumulated in bone marrow from lupus patients but not from nonautoimmune patients undergoing myeloablation, where they were efficiently removed by macrophages (MΦ). Impaired apoptotic cell uptake by MΦ also was seen in mice treated i.p. with pristane (develop lupus) but not mineral oil (MO) (do not develop lupus). The inflammatory response to both pristane and MO rapidly depleted resident (Tim4+) large peritoneal MΦ. The peritoneal exudate of pristane-treated mice contained mainly Ly6Chi inflammatory monocytes; whereas in MO-treated mice, it consisted predominantly of a novel subset of highly phagocytic MΦ resembling small peritoneal MΦ (SPM) that expressed CD138+ and the scavenger receptor Marco. Treatment with anti-Marco-neutralizing Abs and the class A scavenger receptor antagonist polyinosinic acid inhibited phagocytosis of apoptotic cells by CD138+ MΦ. CD138+ MΦ expressed IL-10R, CD206, and CCR2 but little TNF-α or CX3CR1. They also expressed high levels of activated CREB, a transcription factor implicated in generating alternatively activated MΦ. Similar cells were identified in the spleen and lung of MO-treated mice and also were induced by LPS. We conclude that highly phagocytic, CD138+ SPM-like cells with an anti-inflammatory phenotype may promote the resolution of inflammation in lupus and infectious diseases. These SPM-like cells are not restricted to the peritoneum and may help clear apoptotic cells from tissues such as the lung, helping to prevent chronic inflammation.


Assuntos
Lúpus Eritematoso Sistêmico/imunologia , Macrófagos Peritoneais/imunologia , Fagocitose , Sindecana-1/imunologia , Animais , Antígenos Ly/análise , Apoptose , Células da Medula Óssea/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Humanos , Inflamação/imunologia , Subunidade alfa de Receptor de Interleucina-10/genética , Subunidade alfa de Receptor de Interleucina-10/imunologia , Pulmão/citologia , Pulmão/imunologia , Lúpus Eritematoso Sistêmico/induzido quimicamente , Lúpus Eritematoso Sistêmico/fisiopatologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Óleo Mineral/farmacologia , Poli I/farmacologia , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Baço/citologia , Baço/imunologia , Sindecana-1/genética , Terpenos/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
12.
Int J Nanomedicine ; 11: 5897-5904, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28115843

RESUMO

In the present study, we report the interaction of an artificial oligolysine (referred to as AOL) realized in our laboratory with targets of biomedical importance. These included polyinosinic acid (poly rI) and its complex with polycytidylic acid (poly I:C), RNAs with well-known interferon-inducing ability, and double-stranded (ds) DNA. The ability of the peptide to bind both single-stranded poly rI and ds poly I:C RNAs emerged from our circular dichroism (CD) and ultraviolet (UV) studies. In addition, we found that AOL forms complexes with dsDNA, as shown by spectroscopic binding assays and UV thermal denaturation experiments. These findings are encouraging for the possible use of AOL in biomedicine for nucleic acid targeting and oligonucleotide condensation, with the latter being a key step preceding their clinical application. Moreover, we tested the ability of AOL to bind to proteins, using serum albumin as a model protein. We demonstrated the oligolysine-protein binding by CD experiments which suggested that AOL, positively charged under physiological conditions, binds to the protein regions rich in anionic residues. Finally, the morphology characterization of the solid oligolysine, performed by scanning electron microscopy, showed different crystal forms including cubic-shaped crystals confirming the high purity of AOL.


Assuntos
Ácidos Nucleicos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Dicroísmo Circular , DNA/química , DNA/metabolismo , Lisina , Ácidos Nucleicos/química , Poli I/química , Poli I/metabolismo , Poli I-C , Ligação Proteica , Desnaturação Proteica , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Raios Ultravioleta
13.
J Phys Chem B ; 118(43): 12360-5, 2014 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-25280235

RESUMO

Metallization of single-stranded polyinosinic acid (polyI) by Zn(2+) ions at pH 7.0 was studied by differential UV spectroscopy at different temperatures. It was found that polyI is metallized at N7 and N1 atoms of hypoxanthine. The concentration dependence of the degree of binding of Zn(2+) ions to both N7 and N1 sites was obtained, and the corresponding binding constants were determined. Metallization of N1 occurs due to Zn(2+) substituting the imino protons and is effective not only at alkaline but also at neutral pH. This makes multistranded polyI-based systems more promising candidates for use in nanoelectronics than natural DNA sequences, metallization of which can be achieved only at alkaline pH.


Assuntos
Poli I/química , Zinco/química , Concentração de Íons de Hidrogênio , Polimerização , Prótons , Soluções , Espectrofotometria Ultravioleta , Temperatura
14.
PLoS One ; 9(10): e110425, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25343355

RESUMO

Invocation of cellular immunity by epitopic peptides remains largely dependent on empirically developed protocols, such as interfusion of aluminum salts or emulsification using terpenoids and surfactants. To explore novel vaccine formulation, epitopic peptide motifs were co-programmed with structural motifs to produce artificial antigens using our "motif-programming" approach. As a proof of concept, we used an ovalbumin (OVA) system and prepared an artificial protein library by combinatorially polymerizing MHC class I and II sequences from OVA along with a sequence that tends to form secondary structures. The purified endotoxin-free proteins were then examined for their ability to activate OVA-specific T-cell hybridoma cells after being processed within dendritic cells. One clone, F37A (containing three MHC I and two MHC II OVA epitopes), possessed a greater ability to evoke cellular immunity than the native OVA or the other artificial antigens. The sensitivity profiles of drugs that interfered with the F37A uptake differed from those of the other artificial proteins and OVA, suggesting that alteration of the cross-presentation pathway is responsible for the enhanced immunogenicity. Moreover, F37A, but not an epitopic peptide, invoked cellular immunity when injected together with monophosphoryl lipid A (MPL), and retarded tumor growth in mice. Thus, an artificially synthesized protein antigen induced cellular immunity in vivo in the absence of incomplete Freund's adjuvant or aluminum salts. The method described here could be potentially used for developing vaccines for such intractable ailments as AIDS, malaria and cancer, ailments in which cellular immunity likely play a crucial role in prevention and treatment.


Assuntos
Técnicas de Química Combinatória , Epitopos/imunologia , Imunidade Celular/imunologia , Peptídeos/imunologia , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Apresentação do Antígeno/efeitos dos fármacos , Apresentação do Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Proliferação de Células/efeitos dos fármacos , Células Clonais , Apresentação Cruzada/efeitos dos fármacos , Apresentação Cruzada/imunologia , Epitopos/química , Epitopos/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Dados de Sequência Molecular , Ovalbumina/imunologia , Peptídeos/química , Poli I/farmacologia , Polissacarídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequências Repetitivas de Aminoácidos , Receptores Depuradores Classe A/metabolismo , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia
15.
J Immunol ; 193(5): 2416-26, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25057007

RESUMO

Dendritic cells are major APCs that can efficiently prime immune responses. However, the roles of skin-resident Langerhans cells (LCs) in eliciting immune responses have not been fully understood. In this study, we demonstrate for the first time, to our knowledge, that LCs in cynomolgus macaque skin are capable of inducing antiviral-specific immune responses in vivo. Targeting HIV-Gag or influenza hemagglutinin Ags to skin LCs using recombinant fusion proteins of anti-Langerin Ab and Ags resulted in the induction of the viral Ag-specific responses. We further demonstrated that such Ag-specific immune responses elicited by skin LCs were greatly enhanced by TLR ligands, polyriboinosinic polyribocytidylic acid, and R848. These enhancements were not due to the direct actions of TLR ligands on LCs, but mainly dependent on TNF-α secreted from macrophages and neutrophils recruited to local tissues. Skin LC activation and migration out of the epidermis are associated with macrophage and neutrophil infiltration into the tissues. More importantly, blocking TNF-α abrogated the activation and migration of skin LCs. This study highlights that the cross-talk between innate immune cells in local tissues is an important component for the establishment of adaptive immunity. Understanding the importance of local immune networks will help us to design new and effective vaccines against microbial pathogens.


Assuntos
HIV-1/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/farmacologia , Vírus da Influenza A/imunologia , Células de Langerhans/imunologia , Pele/imunologia , Fator de Necrose Tumoral alfa/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/farmacologia , Imunidade Adaptativa/efeitos dos fármacos , Imunidade Adaptativa/fisiologia , Animais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imidazóis/farmacologia , Macaca mulatta , Macrófagos/imunologia , Neutrófilos/imunologia , Poli I/farmacologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
16.
J Photochem Photobiol B ; 134: 64-74, 2014 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-24792476

RESUMO

Studies on the molecular aspects of alkaloid-RNA complexation are of prime importance for the development of rational RNA targeted drug design strategies. Towards this goal, the binding aspects of three novel 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs to four single stranded ribonucleotides, poly(G), poly(I), poly(C) and poly(U), were studied for the first time employing multifaceted biophysical tools. Absorbance and fluorescence studies revealed that these analogs bound non-cooperatively to poly(G) and poly(I) with binding affinities remarkably higher than berberine. The binding of these analogs to poly(U) and poly(C) was weaker in comparison to poly(G) and poly(I) but were one order higher in comparison to berberine. Quantum efficiency values revealed that energy transfer occurred from the RNA bases to the analogs upon complexation. The binding was dominated by large positive entropic contributions and small but favorable enthalpic contributions. Salt dependent studies established that the binding was dominated by hydrophobic forces that contributed around 90% of the total standard molar Gibbs energy. The chain length of the substitution at the 9-position was found to be critical in modulating the binding affinities. These results provide new insights into the binding efficacy of these novel berberine analogs to single stranded RNA sequences.


Assuntos
Berberina/análogos & derivados , Ribonucleotídeos/química , Berberina/metabolismo , Sítios de Ligação , Calorimetria , Dicroísmo Circular , Conformação de Ácido Nucleico , Concentração Osmolar , Poli C/química , Poli C/metabolismo , Poli G/química , Poli G/metabolismo , Poli I/química , Poli I/metabolismo , Poli U/química , Poli U/metabolismo , Teoria Quântica , RNA/química , RNA/metabolismo , Ribonucleotídeos/metabolismo , Espectrometria de Fluorescência , Temperatura , Termodinâmica
17.
Insect Mol Biol ; 23(3): 320-9, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24528536

RESUMO

RNA interference (RNAi) has become a widely used loss-of-function tool in eukaryotes; however, the delivery of double-stranded (ds)RNA) to the target cells remains a major challenge when exploiting the RNAi-technology. In insects, the efficiency of RNAi is highly species-dependent. Yet, the mechanism of cell entry in insects has only been characterized in a cell line of the fruit fly, Drosophila melanogaster, a species that is well known to be poorly amenable to environmental RNAi. In the present paper, we demonstrate that silencing vacuolar H-ATPase 16 (vha16) and clathrin heavy chain (clath), two components of the Clathrin-dependent endocytosis pathway, together with pharmacological inhibition of scavenger receptors with polyinosine and dextran sulphate, can significantly attenuate the highly robust RNAi response in the desert locust, Schistocerca gregaria.


Assuntos
Gafanhotos/genética , Interferência de RNA/efeitos dos fármacos , Animais , Clatrina , Sulfato de Dextrana , Endocitose , Poli I , RNA de Cadeia Dupla/metabolismo , Receptores Depuradores
18.
Hum Gene Ther ; 24(9): 807-13, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24010701

RESUMO

Adeno-associated virus serotype 8 (AAV8) has been demonstrated to be effective for liver-directed gene therapy in humans. Although hepatocytes are the main target cell for AAV8, there is a loss of the viral vector because of uptake by macrophages and Kupffer cells. Reducing this loss would increase the efficacy of viral gene therapy and allow a dose reduction. The receptor mediating this uptake has not been identified; a potential candidate seems the macrophage scavenger receptor A (SR-A) that is involved in the endocytosis of, for instance, adenovirus. In this study we show that SR-A can mediate scAAV8 endocytosis and that blocking it with polyinosinic acid (poly[i]) reduces endocytosis significantly in vitro. Subsequently, we demonstrate that blocking this receptor improves scAAV-mediated liver-directed gene therapy in a model for inherited hyperbilirubinemia, the uridine diphospho-glucuronyl transferase 1A1-deficient Gunn rat. In male rats, preadministration of poly[i] increases the efficacy of a low dose (1×10¹¹ gc/kg) but not of a higher dose (3×10¹¹ gc/kg) scAAV8-LP1-UT1A1. Administration of poly[i] just before the vector significantly increases the correction of serum bilirubin in female rats. In these, the effect of poly[i] is seen by both doses but is more pronounced in the females receiving the low vector, where it also results in a significant increase of bilirubin glucuronides in bile. In conclusion, this study shows that SR-A mediates the endocytosis of AAV8 in vitro and in vivo and that blocking this receptor can improve the efficacy of AAV-mediated liver-directed gene therapy.


Assuntos
Dependovirus/imunologia , Endocitose/efeitos dos fármacos , Macrófagos do Fígado/imunologia , Poli I/metabolismo , Receptores Depuradores Classe A/antagonistas & inibidores , Animais , Bilirrubina/sangue , Células CHO , Linhagem Celular , Cricetulus , Síndrome de Crigler-Najjar/genética , Síndrome de Crigler-Najjar/terapia , Modelos Animais de Doenças , Feminino , Terapia Genética/métodos , Vetores Genéticos , Glucuronosiltransferase/genética , Células HEK293 , Hepatócitos/virologia , Humanos , Macrófagos do Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/metabolismo , Masculino , Ratos , Receptores Depuradores Classe A/efeitos dos fármacos , Receptores Depuradores Classe A/metabolismo , Transdução Genética
19.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 6): 991-6, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23695243

RESUMO

Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three-dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi-1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7-fold and its cytotoxic properties are enhanced compared with the wild-type enzyme.


Assuntos
Endorribonucleases/química , Endorribonucleases/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Poli I/metabolismo , Bacillus/química , Bacillus/enzimologia , Bacillus/genética , Catálise , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Endorribonucleases/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutagênese , Proteínas Mutantes/metabolismo , Difração de Raios X
20.
Opt Express ; 20(24): 26252-60, 2012 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-23187479

RESUMO

This paper presents the optically controllable light scattering based on dye-doped liquid crystals (DDLCs) in a cell, whose substrates are coated with poly(N-vinylcarbazole) (PVK) films. The optical control mechanism is the light-induced dissolution of PVK in DDLCs, which reforms the disordered LC distribution into multiple and micron-sized LC domains. The induced thermal effect on the process is investigated in detail. Scanning electron microscopy images are obtained to show the surface structures of the produced PVK films. The generated scattering can be switched back to the original one by particular thermally induced phase separation. Results indicate that the light-induced thermal effect and photoisomerization lead to the dissolution of PVK in DDLCs. Finally, scattering mode light shutter with different transmission is successfully achieved by illuminating the cell under various light intensities.


Assuntos
Corantes/química , Cristalização/métodos , Luz , Cristais Líquidos/química , Fotoquímica/métodos , Poli I , Espalhamento de Radiação , Corantes/efeitos da radiação , Desenho de Equipamento , Glicocálix , Temperatura Alta , Humanos , Isomerismo , Lasers , Cristais Líquidos/efeitos da radiação
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