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Aging is associated with functional deficits in the naive T cell compartment, which compromise the generation of de novo immune responses against previously unencountered Ags. The mechanisms that underlie this phenomenon have nonetheless remained unclear. We found that naive CD8+ T cells in elderly humans were prone to apoptosis and proliferated suboptimally in response to stimulation via the TCR. These abnormalities were associated with dysregulated lipid metabolism under homeostatic conditions and enhanced levels of basal activation. Importantly, reversal of the bioenergetic anomalies with lipid-altering drugs, such as rosiglitazone, almost completely restored the Ag responsiveness of naive CD8+ T cells. Interventions that favor lipid catabolism may therefore find utility as adjunctive therapies in the elderly to promote vaccine-induced immunity against targetable cancers and emerging pathogens, such as seasonal influenza viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunocompetência/efeitos dos fármacos , Metabolismo dos Lipídeos , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Linfócitos T CD8-Positivos/metabolismo , COVID-19/imunologia , Vacinas Anticâncer/imunologia , Divisão Celular , Feminino , Fenofibrato/farmacologia , Glucose/metabolismo , Antígeno HLA-A2/imunologia , Humanos , Hipolipemiantes/farmacologia , Hipolipemiantes/uso terapêutico , Influenza Humana/imunologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Ativação Linfocitária , Antígeno MART-1/química , Antígeno MART-1/imunologia , Masculino , Pessoa de Meia-Idade , Neoplasias/imunologia , Fragmentos de Peptídeos/imunologia , Rosiglitazona/farmacologia , Método Simples-Cego , Vacinação , Vacinas Virais/imunologia , Adulto JovemRESUMO
Kaposi's sarcoma (KS) is an angioproliferative tumor showing an increased frequency and aggressiveness in HIV-infected subjects (AIDS-KS), due to the combined effects of inflammatory cytokines (IC), angiogenic factors, and the HIV-1 Tat protein. While the introduction of effective combined antiretroviral regimens greatly improved AIDS-KS incidence and course, it continues to be an incurable disease and the development of new rational targeted therapies is warranted. We used the BKV/Tat transgenic mouse model to evaluate the effects of IC and anti-Tat antibodies (Abs) treatment on KS-like lesions arising in BKV/Tat mice. We demonstrated here that IC-treatment increases the severity and delays the regression of KS-like lesions. Further, anti-Tat Abs reduced KS-like lesion severity developing in IC-treated mice when anti-Tat Abs were administered at an early-stage of lesion development as compared to more advanced lesions. Early anti-Tat Abs treatment also accelerated KS-like lesion regression and reduced the rate of severe-grade lesions. This effect was more evident in the first weeks after Ab treatment, suggesting that a longer treatment with anti-Tat Abs might be even more effective, particularly if administered just after lesion development. Although preliminary, these results are encouraging, and the approach deserves further studies for the development of anti-Tat Ab-based therapies for AIDS-KS. Clinical studies specifically addressing the effect of anti-Tat antibodies in treating AIDS-KS are not yet available. Nevertheless, the effectiveness of anti-Tat antibodies in controlling HIV/AIDS progression, likely due to the neutralization of extracellular Tat activities, is suggested by several cross-sectional and longitudinal clinical studies, indicating that anti-Tat Ab treatment or Tat-based vaccines may be effective to treat AIDS-KS patients or prevent the tumor in individuals at risk.
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Anticorpos/farmacologia , Citocinas/metabolismo , Sarcoma de Kaposi/tratamento farmacológico , Sarcoma de Kaposi/patologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Indutores da Angiogênese/metabolismo , Animais , Antirretrovirais/farmacologia , Modelos Animais de Doenças , HIV-1/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Sarcoma de Kaposi/metabolismoRESUMO
Chitosan (CS) and its derivatives are receiving considerable attention for their great biocompatibility and broad-spectrum activities in many fields. In this work, we aimed to characterize the antimicrobial activity of novel chitosan Schiff bases (CSSB). CS was synthesized by double deacetylation of chitin (Cn) after its extraction from the armors of crustaceans Astacus leptodactylus, and CSSB-1 and CSSB-2 were synthesized by interaction of CS with 4-(2-chloroethyl) benzaldehyde (aldehyde-1) and 4-(bromoethyl) benzaldehyde (aldehyde-2), respectively, at room temperature. The synthesized compounds were characterized by elemental analysis, gel permeation chromatography (GPC), infrared spectroscopy (FTIR), thermogravimetry (TG), and differential scanning calorimetry (DSC). The antimicrobial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria and against yeasts (Candida albicans) was significantly increased due to their higher solubility as compared to unmodified CS opening perspectives for the use of these compounds for antimicrobial prevention in different fields as, for example, food industry, cosmetics, or restoration.
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Anti-Infecciosos , Quitosana , Aldeídos , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Benzaldeídos , Quitosana/química , Testes de Sensibilidade Microbiana , Bases de Schiff/química , Bases de Schiff/farmacologia , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The parathyroid glands play an overall regulatory role in the systemic calcium (Ca(2+)) homeostasis. The purpose of the present study was to demonstrate the presence of the Ca(2+) channels transient receptor potential vanilloid (TRPV) 5 and TRPV6 in human parathyroid glands. Semi-quantitative and quantitative PCR was carried out to evaluate the presence of TRPV5 and TRPV6 mRNAs in sporadic parathyroid adenomas and normal parathyroid glands. Western blot and immunocytochemical assays were used to assess protein expression, cellular localization and time expression in primary cultures from human parathyroid adenoma. TRPV5 and TRPV6 transcripts were then identified both in normal and pathological tissues. Predominant immunoreactive bands were detected at 75-80 kD for both vanilloid channels. These channels co-localized with the calcium-sensing receptor (CASR) on the membrane surface, but immunoreactivity was also detected in the cytosol and around the nuclei. Our data showed that western blotting recorded an increase of protein expression of both channels in adenoma samples compared with normal glands suggesting a potential relation with the cell calcium signalling pathway and the pathological processes of these glands.
Assuntos
Adenoma/patologia , Canais de Cálcio/metabolismo , Transformação Celular Neoplásica/patologia , Hiperparatireoidismo Primário/patologia , Glândulas Paratireoides/metabolismo , Canais de Cátion TRPV/metabolismo , Adenoma/genética , Adenoma/metabolismo , Western Blotting , Cálcio/metabolismo , Canais de Cálcio/genética , Membrana Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Humanos , Hiperparatireoidismo Primário/genética , Hiperparatireoidismo Primário/metabolismo , Técnicas Imunoenzimáticas , Glândulas Paratireoides/citologia , Técnicas de Patch-Clamp , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPV/genéticaRESUMO
Adenoviral and mRNA vaccines encoding the viral spike (S) protein have been deployed globally to contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Older individuals are particularly vulnerable to severe infection, probably reflecting age-related changes in the immune system, which can also compromise vaccine efficacy. It is nonetheless unclear to what extent different vaccine platforms are impacted by immunosenescence. Here, we evaluated S protein-specific immune responses elicited by vaccination with two doses of BNT162b2 or ChAdOx1-S and subsequently boosted with a single dose of BNT162b2 or mRNA-1273, comparing age-stratified participants with no evidence of previous infection with SARS-CoV-2. We found that aging profoundly compromised S protein-specific IgG titers and further limited S protein-specific CD4+ and CD8+ T cell immunity as a probable function of progressive erosion of the naive lymphocyte pool in individuals vaccinated initially with BNT162b2. Our results demonstrate that primary vaccination with ChAdOx1-S and subsequent boosting with BNT162b2 or mRNA-1273 promotes sustained immunological memory in older adults and potentially confers optimal protection against coronavirus disease 2019.
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HIV-1 associated neurocognitive disorders (HAND) are a major complication of HIV-1 infection. The mechanism(s) underlying HAND are not completely understood but, based on in vitro studies, the HIV-1 Tat protein may play an important role. In this study, the effect of prolonged exposure to endogenously produced Tat in the brain was investigated using a tat-transgenic (TT) mouse model constitutively expressing the HIV-1 tat gene. We found that stimulus-evoked glutamate exocytosis in the hippocampus and cortex was significantly increased in TT as compared with wild-type control (CC) mice, while GABA exocytosis was unchanged in the hippocampus and decreased in the cortex. This suggests that Tat generates a latent hyper-excitability state, which favors the detrimental effects of neurotoxic and/or excitotoxic agents. To challenge this idea, TT mice were tested for susceptibility to kainate-induced seizures and neurodegeneration, and found to exhibit significantly greater responses to the convulsant agent than CC mice. These results support the concept that constitutive expression of tat in the brain generates a latent excitatory state, which may increase the negative effects of damaging insults. These events may play a key role in the development of HAND.
Assuntos
Encéfalo/patologia , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/virologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Análise de Variância , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/virologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Produtos do Gene tat/farmacologia , Ácido Caínico/toxicidade , Masculino , Camundongos , Camundongos Transgênicos , Neurotransmissores/metabolismo , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Estatísticas não Paramétricas , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
The increasing life expectancy leads to more older adults suffering from infectious diseases. Vaccines are available against diverse infections such as influenza, pneumococcal disease, herpes zoster and tetanus. However, vaccine acceptance is crucial for optimal preventive effect. The objective of the study is to perform a cross-country analysis of the perceptions and decision-making behaviour of older adults regarding vaccinations and their information needs. Focus groups with older adults were conducted in four countries: France, Hungary, Italy and the Netherlands. Data were analysed using thematic analysis. Demographic characteristics of participants were gathered with a questionnaire. Influenza and tetanus vaccines were commonly known, as was the disease influenza. On the contrary, the awareness of the vaccines against pneumococcal disease and herpes zoster were low. Participants also expressed a need for more information on vaccines, such as possible side effects, contra-indications and duration of protection, emphasizing that information is a condition for decision-making on vaccination. General practitioners were found to be the most important in information provision on vaccines. Perceptions on vaccines, such as effectiveness, side effects and safety, as well as perceptions on infectious diseases, such as severity, susceptibility and experiencing an infectious disease, played a role in the decision-making of older adults on vaccines. More awareness of the information needs among older adults with regard to vaccines should be raised among general practitioners and other healthcare providers. This requires appropriate knowledge about the vaccines among healthcare providers as well as communication skills to meet the information needs of older adults.
Assuntos
Doenças Transmissíveis , Herpes Zoster , Influenza Humana , Infecções Pneumocócicas , Tétano , Vacinas , Humanos , Idoso , Influenza Humana/prevenção & controle , Grupos Focais , Infecções Pneumocócicas/prevenção & controleRESUMO
Age-related changes in the immune system are thought to underlie the vulnerability of elderly individuals to emerging viral diseases, such as coronavirus disease 2019 (COVID-19). In this study, we used a fully validated in vitro approach to determine how age impacts the generation of de novo CD8+ T cell responses against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19. Our data revealed a generalized deficit in the ability of elderly individuals to prime the differentiation of naïve precursors into effector CD8+ T cells defined by the expression of interferon (IFN)-γ and the transcription factor T-bet. As a consequence, there was an age-related decline in the diversity of newly generated CD8+ T cell responses targeting a range of typically immunodominant epitopes derived from SARS-CoV-2, accompanied by an overall reduction in the expression frequency of IFN-γ. These findings have potential implications for the development of new strategies to protect the elderly against COVID-19.
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TMEM16A protein, also known as anoctamin-1, has been recently identified as an essential component of Ca(2+)-activated Cl(-) channels. We previously reported the existence of different TMEM16A isoforms generated by alternative splicing. In the present study, we have determined the functional properties of a minimal TMEM16A protein. This isoform, called TMEM16A(0), has a significantly shortened amino-terminus and lacks three alternative segments localized in the intracellular regions of the protein (total length: 840 amino acids). TMEM16A(0) expression is associated with Ca(2+)-activated Cl(-) channel activity as measured by three different functional assays based on the halide-sensitive yellow fluorescent protein, short-circuit current recordings, and patch-clamp technique. However, compared to a longer isoform, TMEM16(abc) (total length: 982 amino acids), TMEM16A(0) completely lacks voltage-dependent activation. Furthermore, TMEM16A(0) and TMEM16A(abc) have similar but not identical responses to extracellular anion replacement, thus suggesting a difference in ion selectivity and conductance. Our results indicate that TMEM16A(0) has the basic domains required for anion transport and Ca(2+)-sensitivity. However, the absence of alternative segments, which are present in more complex isoforms of TMEM16A, modifies the channel gating and ion transport ability.
Assuntos
Canais de Cloreto/química , Proteínas de Membrana/química , Proteínas de Neoplasias/química , Processamento Alternativo , Anoctamina-1 , Proteínas de Bactérias/metabolismo , Cálcio/química , Cloretos/farmacologia , Células HEK293 , Humanos , Íons , Proteínas Luminescentes/metabolismo , Potenciais da Membrana , Microscopia de Fluorescência/métodos , Técnicas de Patch-Clamp , Conformação Proteica , Isoformas de Proteínas , TransfecçãoRESUMO
Adult vaccination programs are receiving increasing attention however, little is known regarding the impact of age on the maintenance of the immune response. We investigated this issue in the context of a human papillomavirus (HPV) vaccination program collecting real-world data on the durability of humoral immunity in 315 female subjects stratified according to vaccination age (adolescents and adults) and sampled at early or late time points after the last vaccine dose. HPV-specific IgGs, but not memory B cells, were induced and maintained at higher levels in subjects vaccinated during adolescence. Nonetheless, antibody functions waned over time to a similar degree in adolescents and adults. To shed light on this phenomena, we analyzed quantitative and qualitative properties of lymphocytes. Similar biochemical features were observed between B-cell subsets from individuals belonging to the two age groups. Long term humoral responses toward vaccines administered at an earlier age were comparably maintained between adolescents and adults. The percentages of naïve B and CD4+ T cells were significantly higher in adolescents, and the latter directly correlated with IgG titers against 3 out of 4 HPV types. Our results indicate that age-specific HPV vaccine responsiveness is mostly due to quantitative differences of immune cell precursors rather than qualitative defects in B cells. In addition, our results indicate that adults also have a good humoral immunogenic profile, suggesting that their inclusion in catch-up programmes is desirable.
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Ca(+)-dependent Clâ» channels (CaCCs) play a variety of physiological roles in different organs and tissues, including transepithelial Clâ» secretion, smooth muscle contraction, regulation of neuronal excitability, and transduction of sensory stimuli. The recent identification of TMEM16A protein as an important component of CaCCs should allow a better understanding of their physiological role, structure-function relationship, and regulatory mechanisms.
Assuntos
Canais de Cloreto/metabolismo , Cloretos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Anoctamina-1 , Canais de Cloreto/química , Células Epiteliais/metabolismo , Humanos , Cinética , Proteínas de Membrana/química , Miócitos de Músculo Liso/metabolismo , Proteínas de Neoplasias/química , Neoplasias/metabolismo , Neurônios/metabolismo , Conformação Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Tat is an early regulatory protein that plays a major role in human HIV-1 replication and AIDS pathogenesis, and therefore, it represents a key target for the host immune response. In natural infection, however, Abs against Tat are produced only by a small fraction (approximately 20%) of asymptomatic individuals and are rarely seen in progressors, suggesting that Tat may possess properties diverting the adaptive immunity from generating humoral responses. Here we show that a Th1-type T cell response against Tat is predominant over a Th2-type B cell response in natural HIV-1 infection. This is likely due to the capability of Tat to selectively target and very efficiently enter CD1a-expressing monocyte-derived dendritic cells (MDDC), which represent a primary target for the recognition and response to virus Ag. Upon cellular uptake, Tat induces MDDC maturation and Th1-associated cytokines and beta-chemokines production and polarizes the immune response in vitro to the Th1 pattern through the transcriptional activation of TNF-alpha gene expression. This requires the full conservation of Tat transactivation activity since neither MDDC maturation nor TNF-alpha production are found with either an oxidized Tat, which does not enter MDDC, or with a Tat protein mutated in the cysteine-rich region (cys22 Tat), which enters MDDC as the wild-type Tat but is transactivation silent. Consistently with these data, inoculation of monkeys with the native wild-type Tat induced a predominant Th1 response, whereas cys22 Tat generated mostly Th2 responses, therefore providing evidence that Tat induces a predominant Th1 polarized adaptive immune response in the host.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/virologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , HIV-1/imunologia , Células Th1/imunologia , Células Th1/virologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/fisiologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Adulto , Idoso , Animais , Células Cultivadas , Quimiocinas CC/biossíntese , Citocinas/biossíntese , Células Dendríticas/metabolismo , Feminino , Anticorpos Anti-HIV/biossíntese , Infecções por HIV/prevenção & controle , Células HeLa , Humanos , Imunidade Inata , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/virologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Células Th1/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Herpes simplex virus type 1 and 2 (HSV1 and HSV2) are global, widespread human pathogens transmitted by direct contact that cause lifelong, recurrent asymptomatic and painful symptomatic clinical illnesses (cold sores, keratitis, blepharitis, meningitis, encephalitis, genital infections), overt disease and severe sequelae in neonatal and immune-compromised patients, and increased risk of cervical cancer and other sexually transmitted infections, including HIV [...].
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Peptide vaccines incorporating B- and T-cell epitopes have shown promise in the context of various cancers and infections. These vaccines are relatively simple to manufacture, but more immunogenic formulations are considered a priority. We developed tetrabranched derivatives for this purpose based on a novel peptide welding technology (PWT). PWTs provide molecular scaffolds for the efficient synthesis of ultrapure peptide dendrimers, which allow the delivery of multiple ligands within a single macromolecular structure. Peptide vaccines incorporating T-cell epitopes derived from melanoma and B-cell epitopes derived from human immunodeficiency virus, synthesized using this approach, elicited primary immune responses in vitro and in vivo. Subcutaneous administration of the B-cell epitope-based vaccines also elicited more potent humoral responses than subcutaneous administration of the corresponding peptides alone. Highly immunogenic peptide epitope-based vaccines can therefore be generated quickly and easily using a novel PWT.
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Advanced age is associated with severe symptoms and death upon SARS-CoV-2 infection. Virus-specific CD8+ T-cell responses have shown to be protective toward critical COVID-19 manifestations, suggesting that suboptimal cellular immunity may contribute to the age-pattern of the disease. The induction of a CD8+ T-cell response against an emerging pathogen like SARS-CoV-2 relies on the activation of naive T cells. To investigate whether the primary CD8+ T-cell response against this virus is defective in advanced age, we used an in vitro approach to prime SARS-CoV-2-specific naive CD8+ T cells from healthy, unexposed donors of different age groups. Compared to younger adults, older individuals display a poor SARS-CoV-2-specific T-cell priming capacity in terms of both magnitude and quality of the response. In addition, older subjects recognize a lower number of epitopes. Our results implicate that immune aging is associated with altered primary SARS-CoV-2-specific CD8+ T-cell responses.
Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Antígenos Virais/imunologia , Células Cultivadas , ELISPOT , Epitopos de Linfócito T/imunologia , Regulação da Expressão Gênica , Voluntários Saudáveis , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Ativação Linfocitária , Pessoa de Meia-Idade , Peptídeos/imunologia , Adulto JovemRESUMO
Expression of TMEM16A protein is associated with the activity of Ca(2+)-activated Cl(-) channels. TMEM16A primary transcript undergoes alternative splicing. thus resulting in the generation of multiple isoforms. We have determined the pattern of splicing and assessed the functional properties of the corresponding TMEM16A variants. We found three alternative exons, 6b, 13, and 15, coding for segments of 22, 4, and 26 amino acids, respectively, which are differently spliced in human organs. By patch clamp experiments on transfected cells, we found that skipping of exon 6b changes the Ca(2+) sensitivity by nearly 4-fold, resulting in Cl(-) currents requiring lower Ca(2+) concentrations to be activated. At the membrane potential of 80 mV, the apparent half-effective concentration decreases from 350 to 90 nm when the segment corresponding to exon 6b is excluded. Skipping of exon 13 instead strongly reduces the characteristic time-dependent activation observed for Ca(2+)-activated Cl(-) channels at positive membrane potentials. This effect was also obtained by deleting only the second pair of amino acids corresponding to exon 13. Alternative splicing appears as an important mechanism to regulate the voltage and Ca(2+) dependence of the TMEM16A-dependent Cl(-) channels in a tissue-specific manner.
Assuntos
Processamento Alternativo , Perfilação da Expressão Gênica , Ativação do Canal Iônico/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Ânions/metabolismo , Anoctamina-1 , Cálcio/metabolismo , Cálcio/farmacologia , Linhagem Celular , Canais de Cloreto , Relação Dose-Resposta a Droga , Humanos , Ativação do Canal Iônico/fisiologia , Transporte de Íons , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Microscopia de Fluorescência , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Técnicas de Patch-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , TransfecçãoRESUMO
Genetic vaccines are safe cost-effective approaches to immunization but DNA immunization is an inefficient process. There is, therefore, a pressing need for adjuvants capable of enhancing the immunogenicity and effectiveness of these vaccines. This is particularly important for diseases for which successful vaccines are still lacking, such as cancer and infectious diseases including HIV-1/AIDS. Here we report an approach to enhance the immunogenicity of DNA vaccines involving the use of transcription factors of the Interferon regulatory factor (IRF) family, specifically IRF-1, IRF-3, and IRF-7 using the tat gene as model antigen. Balb/c mice were immunized by three intramuscular inoculations, using a DNA prime-protein boost protocol, with a DNA encoding tat of HIV-1 and the indicated IRFs and immune responses were compared to those induced by vaccination with tat DNA alone. In vivo administration of plasmid DNA encoding IRF-1, or a mutated version of IRF-1 deleted of the DNA-binding domain, enhanced Tat-specific immune responses and shifted them towards a predominant T helper 1-type immune response with increased IFN-gamma production and cytotoxic T lymphocytes responses. Conversely, the use of IRF-3 or IRF-7 did not affect the tat-induced responses. These findings define IRF-1 and its mutated form as efficacious T helper 1-inducing adjuvants in the context of tat-based vaccination and also providing a new promising candidate for genetic vaccine development.
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Adjuvantes Imunológicos , Fator Regulador 1 de Interferon/imunologia , Vacinas de DNA/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Vacinas contra a AIDS/imunologia , Animais , Linhagem Celular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Feminino , HIV-1/imunologia , Humanos , Imunização , Fator Regulador 1 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Camundongos , Linfócitos T Citotóxicos/imunologiaRESUMO
For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2'-O-methyl-phosphorothioate (2'OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine.
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Distrofina/metabolismo , Nanopartículas/química , Oligorribonucleotídeos Antissenso/fisiologia , Polimetil Metacrilato/química , Animais , Western Blotting , Distrofina/genética , Eletroforese em Gel de Poliacrilamida , Éxons/genética , Terapia Genética/métodos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos mdx , Camundongos Mutantes , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/terapia , Oligorribonucleotídeos Antissenso/genética , Oligorribonucleotídeos Antissenso/metabolismo , Polimetil Metacrilato/síntese químicaRESUMO
The development of therapeutic strategies to control the reactivation of the Herpes Simplex Virus (HSV) is an unaddressed priority. In this study, we evaluated whether Tat, a HIV-1 protein displaying adjuvant functions, could improve previously established HSV-specific memory responses and prevent viral reactivation. To this aim, mice were infected with non-lethal doses of HSV-1 and, 44 days later, injected or not with Tat. Mice were then monitored to check their health status and measure memory HSV-specific cellular and humoral responses. The appearance of symptoms associated with HSV-reactivation was observed at significantly higher frequencies in the control group than in the Tat-treated mice. In addition, the control animals experienced a time-dependent decrease in HSV-specific Immunoglobulin G (IgG), while the Tat-treated mice maintained antibody titers over time. IgG levels were directly correlated with the number of HSV-specific CD8+ T cells, suggesting an effect of Tat on both arms of the adaptive immunity. Consistent with the maintenance of HSV-specific immune memory, Tat-treated mice showed a better control of HSV-1 re-infection. Although further studies are necessary to assess whether similar effects are observed in other models, these results indicate that Tat exerts a therapeutic effect against latent HSV-1 infection and re-infection by favoring the maintenance of adaptive immunity.
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BACKGROUND: Retroviral transduction of cells is improved upon virus adsorption onto immobilized fibronectin (FN) fragments. Because HIV-1 Tat possesses the same functional domains that lead to increased transduction efficiency in FN by colocalization of bound virus and cells, we hypothesized that Tat could enhance gene transfer by a similar mechanism. METHODS: Single-cycle replication retro- or lentivirus carrying green fluorescent protein or cloramphenicol acetyltransferase as reporter genes were added to wells coated with Tat or Tat peptides. Wells were extensively washed to remove unbound virus and levels of transduction were detected by measuring reporter gene expression. Virus adsorption to immobilized Tat was measured using a p24 antigen capture assay. RESULTS: Immobilized Tat efficiently binds retro- and lentiviral particles and mediates virus transmission at virus input doses that were otherwise unable to transduce susceptible cells. Virus adsorption to Tat is not mediated by envelope glycoprotein (Env) because immobilized Tat binds and retains vesicular stomatitis virus G (VSV-G) pseudotypes as well as envelope-free particles. HIV-1 Env or VSV-G are required for Tat-assisted transduction, which is abrogated by an antibody blocking the HIV-1 Env-CD4 interaction. Tat-assisted transduction is mediated by the cysteine-rich region of Tat, which is known to be essential for Tat transactivation activity. However, Tat transactivation is not required for Tat-assisted transduction, as indicated by the enhancement of transduction by transactivation-silent Tat mutants. CONCLUSIONS: Immobilized Tat promotes virus transduction by a transactiva- tion-independent mechanism, which requires binding of virus to Tat. Recombinant Tat or Tat fragments provide a new method to increase efficiency of retro- and lentiviral based gene transfer and gene therapy.