Avaliação da ação antitumoral do complexo natural altamente diluído M1 em células de melanoma humano 1205LU

Orientadora: Profa. Dra. Dorly de Freitas BuchiCoorientadora: Profa. Dra. Carolina Camargo de OliveiraTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa : Curitiba, 26/02/2013Inclui referênciasResumo: O melanoma metastático é a forma mais agressiva de câncer de pele, sendo que aproximadamente 10% dos indivíduos apresentam sobrevida de até 5 anos após o estabelecimento de metástases. Ainda, as abordagens de tratamento envolvendo quimio e radioterapia têm se mostrado pouco eficazes além de efeitos adversos graves. Estudos prévios do Laboratório de células neoplásicas e inflamatórias da Universidade Federal do Paraná utilizando complexos naturais altamente diluídos provenientes de extratos naturais, demonstraram que tais complexos têm efeito modulatório sobre células do sistema imunitário relacionadas com diversos processos celulares importantes na abordagem anticâncer. Uma vez evidenciado, em modelo murino, que tais compostos mostram-se eficazes na promoção de alterações no perfil de expressão gênica, produção de citocinas e alterações morfológicase também na modulação da atividade de células mononucleares no sentido de uma melhor resposta frente a um quadro tumoral, o presente estudo teve como objetivo avaliar o efeito do tratamento in vitro com o complexo natural altamente diluído M1 sobre a linhagem de melanoma metastáticas humano 1205Lu, assim como testar a influência imunitária do tratamento num modelo de cocultura envolvendo células mononucleares humanas obtidas a partir de filtros de leucorredução (câmaras LRS) e células de melanoma 1205Lu. Os resultados mostraram que as células mononucleares previamente tratadas (in vitro) com o complexo natural altamente diluído M1 e posteriormente submetidas à cocultura, promoveram nas células 1205Lu uma redução na taxa de proliferação celular, indução da parada do ciclo celular, aumento da apoptose celular e redução significativa na produção de espécies reativas de oxigênio e nitrogênio. Além disso, redução no potencial invasivo e uma menor expressão de beta-catenina pelas células 1205Lu foi também observada. Quanto às células mononucleares, um aumento na população de células Natural Killer (NK) CD3- /CD56+ foi observado no grupo tratado. Ainda, para células NK isoladas a partir da cultura de células mononucleares e cocultivadas com células 1205Lu, o grupo tratado com o complexo M1 revelou um aumento significativo na citotoxicidade celular frente as células de melanoma. Portanto, o tratamento prévio com o complexo natural altamente diluído M1 demonstrou ser uma abordagem eficaz, não tóxica e barata para melhorar importantes efeitos antitumorais na população de células mononucleares humanas contra o melanoma.Abstract: Metastatic melanoma is the most aggressive form of skin cancer, and the most rapidly expanding cancer in terms of worldwide incidence with a 5 years survival rate of less than 10% after metastasis establishment. Besides that, chemo and radiotherapeutic approaches in melanoma treatment have been shown disappointing results, also resulting in severe adverse effects. Previous studies conducted in Laboratory of Inflammatory and Neoplasic Cells from Universidade Federal do Paraná using murine models, have demonstrated that highly diluted natural complex were able to induce: macrophage activation; an increase in nitric oxide production; and increase in CD45R+ cells in bone marrow, a decrease in tumor growth in Sarcoma 180 in vivo model and an increase in tumoricidal response of activated lymphocytes against B16F10 melanoma cells in vitro. Here, we performed assays to evaluate in vitro effects of highly diluted natural complex, M1, in human metastatic melanoma cell lineage 1205Lu. Also, we have tested the treatment immunological influence in a coculture model between human mononuclear cells obtained from lekoreduction chambers (LRS chambers) after plateletpheresis procedure, and melanoma cells (1205Lu). Results have shown that mononuclear cells previously treated (in vitro) for 48 hours with M1 and then submitted to coculture, promoted a reduction in proliferation rate a, induced cell cycle arrest and a increased cell apoptosis in 1205Lu cells. Also, a significant reduction in nitrogen and oxygen reactive species production was seen in tumoral cells. Combined to those results, a reduction in cell invasion and a lower betacatenin expression was also observed. Besides that, when mononuclear cells were analyzed by flow cytometry an increase in CD3-/CD56+ Natural Killer cell population was seen in treated group. Finally, when isolated from mononuclear cultures, and co- cultured with 1205Lu, Natural Killer cells from M1 treated group, revealed a significant increased citotoxicity against melanoma cells. In conclusion, pre treatment with the highly diluted natural complex M1 demonstrated to be an effective and non-toxic approach to improve important anti tumoral effects in human mononuclear cell population against melanoma

Why not "do simple things in a simple way": Use of the Pap test as the first step in screening genetic stability for human cultured stem cell therapy?

The aim of this study was to analyze adipose tissue-derived mesenchymal stem cells (AT-MSCs) using the Pap test as a first screening step to evaluate genetic stability. Human adipose tissue from six healthy female donors was obtained from elective liposuction procedures. The cells were isolated, cultivated at P2/P3, characterized by flow cytometric analysis, and differentiation induced. The AT-MSCs were stained by Papanicolaou staining and analyzed according to the Bethesda classification, and viability-apoptosis relationships were evaluated. The results of the Pap test for Sample I indicated high-grade alterations consistent with genetic instability; for Samples II-V, atypical cells of undetermined significance; and for Sample VI, normal cells. These results demonstrate the potential of using the Pap test as an initial screening step to evaluate the genetic stability of cultured AT-MSCs and also suggest its use for other adherent cells such as embryonic stem cells or induced pluripotent stem cells

Developments on drug discovery and on new therapeutics: highly diluted tinctures act as biological response modifiers

<p>Abstract</p> <p>Background</p> <p>In the search for new therapies novel drugs and medications are being discovered, developed and tested in laboratories. Highly diluted substances are intended to enhance immune system responses resulting in reduced frequency of various diseases, and often present no risk of serious side-effects due to its low toxicity. Over the past years our research group has been investigating the action of highly diluted substances and tinctures on cells from the immune system.</p> <p>Methods</p> <p>We have developed and tested several highly diluted tinctures and here we describe the biological activity of M1, M2, and M8 both <it>in vitro </it>in immune cells from mice and human, and <it>in vivo </it>in mice. Cytotoxicity, cytokines released and NF-κB activation were determined after <it>in vitro </it>treatment. Cell viability, oxidative response, lipid peroxidation, bone marrow and lymph node cells immunophenotyping were accessed after mice <it>in vivo </it>treatment.</p> <p>Results</p> <p>None of the highly diluted tinctures tested were cytotoxic to macrophages or K562. Lipopolysaccharide (LPS)-stimulated macrophages treated with all highly diluted tinctures decreased tumour necrosis factor alpha (TNF-α) release and M1, and M8 decreased IFN-<it>γ </it>production. M1 has decreased NF-κB activity on TNF-α stimulated reporter cell line. <it>In vivo </it>treatment lead to a decrease in reactive oxygen species (ROS), nitric oxide (NO) production was increased by M1, and M8, and lipid peroxidation was induced by M1, and M2. All compounds enhanced the innate immunity, but M1 also augmented acquired immunity and M2 diminished B lymphocytes, responsible to acquired immunity.</p> <p>Conclusions</p> <p>Based on the results presented here, these highly diluted tinctures were shown to modulate immune responses. Even though further investigation is needed there is an indication that these highly diluted tinctures could be used as therapeutic interventions in disorders where the immune system is compromised.</p

Suplementação cronica da dieta com B-hidroxi B-metilbutirato (HMB) e treinamento contra-resistido reduzem a taxa de crescimento do tumor de Walker 256 : identificação dos mediadores participantes neste processo

Orientador : Prof. Dr. Luiz Claudio FernandesDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 2007Inclui bibliografiaVários estudos têm mostrado que a suplementação tanto com o aminoácido Leucina, quanto com seu metabólito, o ß-hidróxi-ß-metilbutirato (HMB), leva a incremento dos parâmetros imunitários. Além disso, o HMB reduz o quadro de hipercatabolismo presente na caquexia em pacientes com câncer. A realização de exercício físico moderado tem sido demonstrado ser útil em amenizar o quadro hipercatabólico, bem como melhorar as funções imunitárias dos indivíduos com câncer. O objetivo deste estudo foi investigar a associação do exercício físico contra-resistido com a suplementação com HMB em ratos portadores do tumor de Walker 256. Ratos Wistar, 70 dias de idade, foram suplementados com HMB (76 mg/kg/dia) por gavagem e submetidos a treinamento físico que consistiu na realização de 10 séries de saltos (com sobrecarga relativa a 50% do peso corporal acoplada ao tronco) por dia, com duração de 30 segundos cada e intervalos de 1 minuto entre as séries, 4 vezes por semana durante 8 semanas. Após 6 semanas treinamento foi injetado 3x10 elevado a 7 células de suspensão de células do tumor de Walker 256 (1mL) nos ratos onde estabeleceu-se os grupos a seguir: Sedentário portador de tumor (SW), sedentário portador de tumor suplementado com HMB (SHW), Exercitado portador de tumor (EXW) e Exercitado portador de tumor suplementado com HMB (EXHW). Duas semanas a pós a inoculação do tumor no flanco direito os animais foram ortotanasiados, o tumor removido, pesado e as células submetidas a estudo quanto à proliferação celular pela incorporação de (2 elevado a-14C)-timidina em DNA, apoptose utilizando anexina-V-FITC em citometria de fluxo, a expressão das proteínas Bcl-2, Bax, subunidades P65 e P52 do fator de transcrição nuclear kB, proteína inibitória deste fator do NF-kB (IkB) e fator de indução de proteólise (PIF) foram determinadas por western blotting. A massa tumoral dos animais dos grupos exercitados (EXW) e exercitados e suplementados com HMB (EXHW) foi significativamente menor quando comparada à dos demais grupos (p<0,05). A proliferação celular das células destes mesmos grupos, em cultura, também foi menor e houve efeito aditivo do treinamento e suplementação (p<0,05). As células tumorais dos animais exercitados apresentaram maior taxa de apoptose (p<0,05) que à do grupo controle (SW). A suplementação com HMB não induziu a alteração significativa na expressão da Bcl2. Por outro lado, a atividade física foi hábil em promover redução em 40,5% na expressão da Bcl2 no grupo EXW (p<0,05 vs. SW e SHW). A associação da atividade física com a suplementação incrementou esta redução para 58,5% quando comparada à dos grupos sedentários (p<0,01 vs. SW e SHW). Quanto à Bax, a atividade física induziu a um aumento de 1,7 vezes na sua expressão quando comparada à do grupo SW (p<0,05), mas não foi diferente quando comparada à do SHW (p>0,05). A associação da atividade física e suplementação com HMB elevou a expressão da Bax em 2,5 vezes quando comparada à SW (p<0,05), e em 1,7 vezes quando comparada à do SHW (p<0,05). A suplementação com HMB reduziu a expressão da p65 em 1,5 vezes (p<0,05 vs. SW). A atividade física levou a uma redução de 1,75 vezes que foi significativa quando comparada à do SW (p<0,01), mas não em relação à do SHW (p>0,05). A associação da atividade física e suplementação reduziu a expressão em 6 vezes quando comparada à do SW (p<0,001), em 4 vezes à do SHW (p<0,05) e em 3,4 vezes à do EXW (p<0,05). A expressão da subunidade P52 não mostrou ser significativamente diferente entre os grupos (p>0,05). A suplementação com HMB aumentou a expressão da IkB em 2,3 vezes (p<0,05 vs. SW). A atividade física promoveu o mesmo efeito da suplementação com HMB (p<0,05 vs SW). A associação da atividade física e suplementação com HMB tiveram efeito aditivo sobre a expressão da Ikb, elevando para 3,3 vezes quando comparada à do SW (p<0,001) e em 1,4 vezes quando comparada à dos SHW e EXW (p<0,05). Quanto ao fator de indução de proteólise (PIF), a suplementação com HMB não modificou sua expressão (p>0,05 vs. SW). A atividade física foi hábil em reduzir a expressão do PIF em 1,84 vezes quando comparada à dos grupos SW e SHW (p<0,05). A associação da atividade física e suplementação com HMB foi hábil em reduzir para 2,75 vezes (p<0,001 vs. SW e SHW). Quando comparado com a atividade física houve redução de 1,5 vezes (p<0,05 vs. EXW). Estes resultados sugerem que a associação da suplementação com HMB e exercício físico contraresistido reduz a massa tumoral e nos mecanismos envolvidos estão a redução na expressão de proteínas envolvidas no processo de proliferação celular, tais como a subunidade p65 do fator de transcrição NF-kB e incremento da expressão da proteína inibitória deste fator (IkB), aumento da expressão da proteína pró-apoptótica Bax e diminuição da anti-apoptótica Bcl-2 nos grupos exercitados, o que sinaliza para um aumento no balanço pró-apoptótico do tecido tumoral. A proteína PIF, importante fator relacionado com a instalação do quadro caquético, também mostrou ter sua expressão diminuída nos grupos exercitados.Many studies have shown that leucine supplementation or with ß-hidroxi-ß- methyllbutyrate (HMB) increases immune response. In addition, HMB is able to reduce cachexia in cancer patients. Moderate physical activity also has been shown be able to reduce hypercatabolism in cancer patients. The main goal of this study is to investigate the effect of the association of HMB supplementation and physical activity in Walker 256 tumor-bearing rats. Rats 70 days old were HMB supplemented (76 mg/kg/day) by gavage and trained during 8 weeks. Jump training consisted by ten sets of 10 jumps in water tank in 30 seconds, with an overload attached to the body equals to 50% of the body mass, followed by 1 minute o resting, four times per week. At the seventh week of training were inoculated in some rats a suspension of Walker tumor cels (3x107/ml) getting the groups: tumor-bearing (SW), tumor-bearing HMB supplemented (SHW), exercised (EXW) and exercised supplemented (EXHW). After 2 weeks the rats were killed and the tumor removed and the were isolated for study of cellular proliferation by (2 to the power of -14C)-thymidine incorporation into DNA, apoptosis using anexin-V measurent by flow cytometer, Bcl-2, Bax, NFêB subunits p65, p52 and iêB and finally proteolysis inducing factor (PIF) by western blotting. Tumor weight in the EXW and EXHW was lower when compared to the other groups (p<0.05). Proliferation rate was also lower in these groups and there was additive effect of training and supplementation (p<0.05). Apoptosis rate was higher in the EXW group (p<0.05 vs. SW). Bcl-2 expression did not change by HMB but the exercise caused a reduction by 40.5% in the EXW (P<0.05 vs. SW and SHW). The association between exercise and HMB supplementation caused further reduction by 58.5% (p<0.01 vs. SW e SHW). Physical activity increased Bax expression by 1.7-fold (p<0.05 vs. SW) but was not different from SHW (p>0.05). Physical activity and supplementation increased Bax expression by 2.5-fold and 1.7-fold when compared to SW and SHW, respectively (p<0.05). HMB supplementation reduced p65 expression by 1.5-fold (p<0,05 vs. SW) and the physical activity by 1.75-fold (p<0.01 vs. SW). The association of both procedures reduced by 6-fold, 4-fold and 3.4-fold when compared to SW (p<0.001), SHW (p<0.05) and EXW (p<0.05), respectively. P52 expression did not change between the groups. HMB increases IkB expression by 2.3-fold (p<0,05 vs. SW), as well as the physical activity (p<0.05 vs SW). Physical activity plus HMB increased Ikb expression by 3.3-fold when compared to SW (p<0.001) and by 1.4-fold to SHW e EXW (p<0.05). PIF expression was reduced in the EXW group by 1.84-fold (p<0.05 vs. SW e SHW). The association with HMB caused further reduction by 2.75-fold (p<0.001 vs. SW e SHW). Our data suggest that HMB supplementation associated to physical exercise reduced tumor growth which was accompanied by the reduction of p65 subunit of NF-kB expression and increase of IkB. Also, there was an increase of Bax and reduction of Bcl-2 expression. PIF expression was reduced in the exercised groups

Characterization of Dendritic Cell-Derived Extracellular Vesicles During Dengue Virus Infection

The dengue virus (DENV), transmitted by Aedes spp. mosquitoes, is one of the most important arboviral infections in the world. Dengue begins as a febrile condition, and in certain patients, it can evolve severe clinical outcomes, such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The reasons why certain patients develop DHF or DSS have not been thoroughly elucidated to date, and both patient and viral factors have been implicated. Previous work has shown that a severe immune dysfunction involving dendritic cells and T cells plays a key role in increasing the disease severity, especially in secondary heterologous infections. Extracellular vesicles (EVs) are membranous particles that are secreted by several cell types involved in homeostatic and pathological processes. Secretion of EVs by infected cells can enhance immune responses or favor viral evasion. In this study, we compare the molecular content of EVs that are secreted by human primary dendritic cells under different conditions: uninfected or infected with DENV3 strains isolated from patients with different infection phenotypes (a severe case involving DSS and a mild case). Human monocyte-derived dendritic cells (mdDCs) were infected with the dengue virus strains DENV3 5532 (severe) or DENV3 290 (mild), and the EVs were isolated. The presence of cup-shaped EVs was confirmed by electron microscopy and immunostaining with CD9, CD81, and CD83. The RNA content from the mdDC-infected cells contained several mRNAs and miRNAs related to immune responses compared to the EVs from mock-infected mdDCs. A number of these RNAs were detected exclusively during infection with DENV3 290 or DENV3 5532. This result suggests that the differential immune modulation of mdDCs by dengue strains can be achieved through the EV pathway. Additionally, we observed an association of EVs with DENV-infectious particles that seem to be protected from antibodies targeting the DENV envelope protein. We also showed that EVs derived from cells treated with IFN alpha have a protective effect against DENV infection in other cells. These results suggested that during DENV infection, the EV pathway could be exploited to favor viral viability, although immune mechanisms to counteract viral infection can also involve DC-derived EVs

Table_1_Characterization of Dendritic Cell-Derived Extracellular Vesicles During Dengue Virus Infection.XLSX

<p>The dengue virus (DENV), transmitted by Aedes spp. mosquitoes, is one of the most important arboviral infections in the world. Dengue begins as a febrile condition, and in certain patients, it can evolve severe clinical outcomes, such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The reasons why certain patients develop DHF or DSS have not been thoroughly elucidated to date, and both patient and viral factors have been implicated. Previous work has shown that a severe immune dysfunction involving dendritic cells and T cells plays a key role in increasing the disease severity, especially in secondary heterologous infections. Extracellular vesicles (EVs) are membranous particles that are secreted by several cell types involved in homeostatic and pathological processes. Secretion of EVs by infected cells can enhance immune responses or favor viral evasion. In this study, we compare the molecular content of EVs that are secreted by human primary dendritic cells under different conditions: uninfected or infected with DENV3 strains isolated from patients with different infection phenotypes (a severe case involving DSS and a mild case). Human monocyte-derived dendritic cells (mdDCs) were infected with the dengue virus strains DENV3 5532 (severe) or DENV3 290 (mild), and the EVs were isolated. The presence of cup-shaped EVs was confirmed by electron microscopy and immunostaining with CD9, CD81, and CD83. The RNA content from the mdDC-infected cells contained several mRNAs and miRNAs related to immune responses compared to the EVs from mock-infected mdDCs. A number of these RNAs were detected exclusively during infection with DENV3 290 or DENV3 5532. This result suggests that the differential immune modulation of mdDCs by dengue strains can be achieved through the EV pathway. Additionally, we observed an association of EVs with DENV-infectious particles that seem to be protected from antibodies targeting the DENV envelope protein. We also showed that EVs derived from cells treated with IFN alpha have a protective effect against DENV infection in other cells. These results suggested that during DENV infection, the EV pathway could be exploited to favor viral viability, although immune mechanisms to counteract viral infection can also involve DC-derived EVs.</p

Table_3_Characterization of Dendritic Cell-Derived Extracellular Vesicles During Dengue Virus Infection.XLSX

<p>The dengue virus (DENV), transmitted by Aedes spp. mosquitoes, is one of the most important arboviral infections in the world. Dengue begins as a febrile condition, and in certain patients, it can evolve severe clinical outcomes, such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The reasons why certain patients develop DHF or DSS have not been thoroughly elucidated to date, and both patient and viral factors have been implicated. Previous work has shown that a severe immune dysfunction involving dendritic cells and T cells plays a key role in increasing the disease severity, especially in secondary heterologous infections. Extracellular vesicles (EVs) are membranous particles that are secreted by several cell types involved in homeostatic and pathological processes. Secretion of EVs by infected cells can enhance immune responses or favor viral evasion. In this study, we compare the molecular content of EVs that are secreted by human primary dendritic cells under different conditions: uninfected or infected with DENV3 strains isolated from patients with different infection phenotypes (a severe case involving DSS and a mild case). Human monocyte-derived dendritic cells (mdDCs) were infected with the dengue virus strains DENV3 5532 (severe) or DENV3 290 (mild), and the EVs were isolated. The presence of cup-shaped EVs was confirmed by electron microscopy and immunostaining with CD9, CD81, and CD83. The RNA content from the mdDC-infected cells contained several mRNAs and miRNAs related to immune responses compared to the EVs from mock-infected mdDCs. A number of these RNAs were detected exclusively during infection with DENV3 290 or DENV3 5532. This result suggests that the differential immune modulation of mdDCs by dengue strains can be achieved through the EV pathway. Additionally, we observed an association of EVs with DENV-infectious particles that seem to be protected from antibodies targeting the DENV envelope protein. We also showed that EVs derived from cells treated with IFN alpha have a protective effect against DENV infection in other cells. These results suggested that during DENV infection, the EV pathway could be exploited to favor viral viability, although immune mechanisms to counteract viral infection can also involve DC-derived EVs.</p

Table_4_Characterization of Dendritic Cell-Derived Extracellular Vesicles During Dengue Virus Infection.XLSX

<p>The dengue virus (DENV), transmitted by Aedes spp. mosquitoes, is one of the most important arboviral infections in the world. Dengue begins as a febrile condition, and in certain patients, it can evolve severe clinical outcomes, such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The reasons why certain patients develop DHF or DSS have not been thoroughly elucidated to date, and both patient and viral factors have been implicated. Previous work has shown that a severe immune dysfunction involving dendritic cells and T cells plays a key role in increasing the disease severity, especially in secondary heterologous infections. Extracellular vesicles (EVs) are membranous particles that are secreted by several cell types involved in homeostatic and pathological processes. Secretion of EVs by infected cells can enhance immune responses or favor viral evasion. In this study, we compare the molecular content of EVs that are secreted by human primary dendritic cells under different conditions: uninfected or infected with DENV3 strains isolated from patients with different infection phenotypes (a severe case involving DSS and a mild case). Human monocyte-derived dendritic cells (mdDCs) were infected with the dengue virus strains DENV3 5532 (severe) or DENV3 290 (mild), and the EVs were isolated. The presence of cup-shaped EVs was confirmed by electron microscopy and immunostaining with CD9, CD81, and CD83. The RNA content from the mdDC-infected cells contained several mRNAs and miRNAs related to immune responses compared to the EVs from mock-infected mdDCs. A number of these RNAs were detected exclusively during infection with DENV3 290 or DENV3 5532. This result suggests that the differential immune modulation of mdDCs by dengue strains can be achieved through the EV pathway. Additionally, we observed an association of EVs with DENV-infectious particles that seem to be protected from antibodies targeting the DENV envelope protein. We also showed that EVs derived from cells treated with IFN alpha have a protective effect against DENV infection in other cells. These results suggested that during DENV infection, the EV pathway could be exploited to favor viral viability, although immune mechanisms to counteract viral infection can also involve DC-derived EVs.</p

Table_2_Characterization of Dendritic Cell-Derived Extracellular Vesicles During Dengue Virus Infection.XLSX

<p>The dengue virus (DENV), transmitted by Aedes spp. mosquitoes, is one of the most important arboviral infections in the world. Dengue begins as a febrile condition, and in certain patients, it can evolve severe clinical outcomes, such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The reasons why certain patients develop DHF or DSS have not been thoroughly elucidated to date, and both patient and viral factors have been implicated. Previous work has shown that a severe immune dysfunction involving dendritic cells and T cells plays a key role in increasing the disease severity, especially in secondary heterologous infections. Extracellular vesicles (EVs) are membranous particles that are secreted by several cell types involved in homeostatic and pathological processes. Secretion of EVs by infected cells can enhance immune responses or favor viral evasion. In this study, we compare the molecular content of EVs that are secreted by human primary dendritic cells under different conditions: uninfected or infected with DENV3 strains isolated from patients with different infection phenotypes (a severe case involving DSS and a mild case). Human monocyte-derived dendritic cells (mdDCs) were infected with the dengue virus strains DENV3 5532 (severe) or DENV3 290 (mild), and the EVs were isolated. The presence of cup-shaped EVs was confirmed by electron microscopy and immunostaining with CD9, CD81, and CD83. The RNA content from the mdDC-infected cells contained several mRNAs and miRNAs related to immune responses compared to the EVs from mock-infected mdDCs. A number of these RNAs were detected exclusively during infection with DENV3 290 or DENV3 5532. This result suggests that the differential immune modulation of mdDCs by dengue strains can be achieved through the EV pathway. Additionally, we observed an association of EVs with DENV-infectious particles that seem to be protected from antibodies targeting the DENV envelope protein. We also showed that EVs derived from cells treated with IFN alpha have a protective effect against DENV infection in other cells. These results suggested that during DENV infection, the EV pathway could be exploited to favor viral viability, although immune mechanisms to counteract viral infection can also involve DC-derived EVs.</p

Table_5_Characterization of Dendritic Cell-Derived Extracellular Vesicles During Dengue Virus Infection.XLSX

<p>The dengue virus (DENV), transmitted by Aedes spp. mosquitoes, is one of the most important arboviral infections in the world. Dengue begins as a febrile condition, and in certain patients, it can evolve severe clinical outcomes, such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The reasons why certain patients develop DHF or DSS have not been thoroughly elucidated to date, and both patient and viral factors have been implicated. Previous work has shown that a severe immune dysfunction involving dendritic cells and T cells plays a key role in increasing the disease severity, especially in secondary heterologous infections. Extracellular vesicles (EVs) are membranous particles that are secreted by several cell types involved in homeostatic and pathological processes. Secretion of EVs by infected cells can enhance immune responses or favor viral evasion. In this study, we compare the molecular content of EVs that are secreted by human primary dendritic cells under different conditions: uninfected or infected with DENV3 strains isolated from patients with different infection phenotypes (a severe case involving DSS and a mild case). Human monocyte-derived dendritic cells (mdDCs) were infected with the dengue virus strains DENV3 5532 (severe) or DENV3 290 (mild), and the EVs were isolated. The presence of cup-shaped EVs was confirmed by electron microscopy and immunostaining with CD9, CD81, and CD83. The RNA content from the mdDC-infected cells contained several mRNAs and miRNAs related to immune responses compared to the EVs from mock-infected mdDCs. A number of these RNAs were detected exclusively during infection with DENV3 290 or DENV3 5532. This result suggests that the differential immune modulation of mdDCs by dengue strains can be achieved through the EV pathway. Additionally, we observed an association of EVs with DENV-infectious particles that seem to be protected from antibodies targeting the DENV envelope protein. We also showed that EVs derived from cells treated with IFN alpha have a protective effect against DENV infection in other cells. These results suggested that during DENV infection, the EV pathway could be exploited to favor viral viability, although immune mechanisms to counteract viral infection can also involve DC-derived EVs.</p