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Nanomedicine (Lond) ; 15(27): 2671-2688, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33112210


Aim: This research aims to identify important formulation parameters for the enhancement of nanoparticle (NP) uptake and decreasing the cytotoxicity in macrophages. Materials & methods: Fluorescent poly(lactic-co-glycolic acid) (PLGA) nanocarriers were characterized for size distributions, zeta potential and encapsulation efficiency. Incubation time, size class, PLGA derivative and chitosan derivative were assessed for uptake kinetics and cell viability. Results: The major determining factor for enhancing cellular uptake were chitosan coatings, combined with acid-terminated PLGA and small NP size. Moreover, cytotoxicity was more favorable for small, chitosan glutamate-coated, acid-terminated PLGA NPs compared with its plain chitosan-coated counterparts. Conclusion: Chitosan glutamate has been shown to be a valuable alternative coating material for acid-terminated PLGA NPs to efficiently and safely target macrophages.

Intervirology ; 62(3-4): 134-144, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31533107


OBJECTIVES: Differences have been observed in the susceptibility of macrophage cell lines to respiratory syncytial virus (RSV) infection. In this study, we evaluated whether the type of macrophage cell line and RSV strain used have an influence on the infectivity and production of progeny virus. METHODS: Both human and murine macrophage-like cell lines were infected with different RSV strains, both lab strains as well as clinical isolates. The infection was evaluated after 24 and 72 h by immunofluorescence staining and microscopic analysis, and the production of new virus particles was determined by plaque assay. RESULTS: Susceptibility of macrophages to RSV was influenced by the RSV strain used but was mostly dependent on the macrophage cell line. Numbers of infected cells and virus production were generally very low or absent in murine cell lines. In human cell lines, clear infection was observed associated with production of new virus particles. CONCLUSION: Differences in susceptibility of macrophage cell lines to RSV infection are primarily related to the species of origin of the cell line but are also influenced by the RSV strain.

Especificidade de Hospedeiro , Macrófagos/virologia , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Replicação Viral , Animais , Linhagem Celular , Humanos , Camundongos , Carga Viral , Ensaio de Placa Viral
Virol J ; 14(1): 157, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28818113


BACKGROUND: Host proteases have been shown to play important roles in many viral activities such as entry, uncoating, viral protein production and disease induction. Therefore, these cellular proteases are putative targets for the development of antivirals that inhibit their activity. Host proteases have been described to play essential roles in Ebola, HCV, HIV and influenza, such that specific protease inhibitors are able to reduce infection. RSV utilizes a host protease in its replication cycle but its potential as antiviral target is unknown. Therefore, we evaluated the effect of protease inhibitors on RSV infection. METHODS: To measure the sensitivity of RSV infection to protease inhibitors, cells were infected with RSV and incubated for 18 h in the presence or absence of the inhibitors. Cells were fixed, stained and studied using fluorescence microscopy. RESULTS: Several protease inhibitors, representing different classes of proteases (AEBSF, Pepstatin A, E-64, TPCK, PMSF and aprotinin), were tested for inhibitory effects on an RSV A2 infection of HEp-2 cells. Different treatment durations, ranging from 1 h prior to inoculation and continuing for 18 h during the assay, were evaluated. Of all the inhibitors tested, AEBSF and TPCK significantly decreased RSV infection. To ascertain that the observed effect of AEBSF was not a specific feature related to HEp-2 cells, A549 and BEAS-2B cells were also used. Similar to HEp-2, an almost complete block in the number of RSV infected cells after 18 h of incubation was observed and the effect was dose-dependent. To gain insight into the mechanism of this inhibition, AEBSF treatment was applied during different phases of an infection cycle (pre-, peri- and post-inoculation treatment). The results from these experiments indicate that AEBSF is mainly active during the early entry phase of RSV. The inhibitory effect was also observed with other RSV isolates A1998/3-2 and A2000/3-4, suggesting that this is a general feature of RSV. CONCLUSION: RSV infection can be inhibited by broad serine protease inhibitors, AEBSF and TPCK. We confirmed that AEBSF inhibition is independent of the cell line used or RSV strain. The time point at which treatment with the inhibitor was most potent, was found to coincide with the expected moment of entry of the virion with the host cell.

Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/patogenicidade , Inibidores de Serino Proteinase/farmacologia , Sulfonas/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Células A549 , Aprotinina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Endopeptidases/efeitos dos fármacos , Humanos , Cinética , Leucina/análogos & derivados , Leucina/antagonistas & inibidores , Pepstatinas/antagonistas & inibidores , Fatores de Tempo , Tosilfenilalanil Clorometil Cetona/antagonistas & inibidores , Proteínas Virais/metabolismo
Immunobiology ; 222(6): 797-806, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27899210


Sialoadhesin (Sn) is a surface receptor expressed on resident macrophages with the ability to bind with sialic acids. During inflammation, an upregulation of Sn is observed. Upon binding of monoclonal antibodies to Sn, the receptor becomes internalized and this has been observed in multiple species. The latter characteristic, combined with the strong upregulation of Sn on inflammatory macrophages and the fact that Sn-positive macrophages contribute to certain inflammatory diseases, makes Sn an interesting entry portal for phenotype-modulating or cytotoxic drugs. Such drugs or toxins can be linked to Sn-specific antibodies which should enable their targeted uptake by macrophages. However, the activity of such drugs depends not only on their internalization but also on the intracellular trafficking and final fate in the endolysosomal system. Although information is available for porcine Sn, the detailed mechanisms of human and mouse Sn internalization and subsequent intracellular trafficking are currently unknown. To allow development of Sn-targeted therapies, differences across species and cellular background need to be characterized in more detail. In the current report, we show that internalization of human and mouse Sn is dynamin-dependent and clathrin-mediated, both in primary macrophages and CHO cell lines expressing a recombinant Sn. In primary macrophages, internalized Sn-specific F(ab')2 fragments are located mostly in the early endosomes. With Fc containing Sn-specific antibodies, there is a slight shift towards lysosomal localization in mouse macrophages, possibly because of an interaction with Fc receptors. Surprisingly, in CHO cell lines expressing Sn, there is a predominant lysosomal localization. Our results show that the mechanism of Sn internalization and intracellular trafficking is concurrent in the tested species. The cellular background in which Sn is expressed and the type of antibody used can affect the intracellular fate, which in turn can impact the activity of antibody-based therapeutic interventions via Sn.

Complexo Antígeno-Anticorpo/metabolismo , Endossomos/metabolismo , Inflamação/metabolismo , Macrófagos/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Células CHO , Clatrina/metabolismo , Cricetulus , Regulação para Baixo , Dinaminas/metabolismo , Endocitose , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transporte Proteico , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Ácidos Siálicos/metabolismo , Suínos
Cell Immunol ; 312: 51-60, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27993350


Sialoadhesin (Sn) is a surface receptor expressed on macrophages in steady state conditions, but during inflammation, Sn can be upregulated both on macrophages and on circulating monocytes. It was shown for different species that Sn becomes internalized after binding with monoclonal antibodies. These features suggest that Sn is a potential target for immunotherapies. In this study, human and mouse macrophages were treated with anti-Sn monoclonal antibodies or F(ab')2 fragments and the effect of their binding to Sn on phagocytosis was analyzed. Binding of antibodies to Sn resulted in delayed and reduced phagocytosis of fluorescent beads. No effect was observed on Fc-mediated phagocytosis or phagocytosis of bacteria by human macrophages. In contrast, an enhanced phagocytosis of bacteria by mouse macrophages was detected. These results showed that stimulation of Sn could have different effects on macrophage phagocytosis, depending both on the type of phagocytosis and cellular background.

Anticorpos Monoclonais/farmacologia , Inflamação/imunologia , Macrófagos/efeitos dos fármacos , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Células Cultivadas , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Especificidade da Espécie
Antibodies (Basel) ; 5(2)2016 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-31557988


Sialoadhesin (Sn) is a surface receptor expressed on a subset of macrophages in steady state conditions. During inflammation and diseases, Sn is highly upregulated on macrophages and blood monocytes. Therefore, therapies using monoclonal antibodies (mAbs) to target Sn-positive (Sn+) cells are a potential strategy for targeted treatment. It has been shown that Sn internalizes after binding with a mAb, though it is not clear whether this is species-specific. In this study, new Sn-specific mAbs were developed and analyzed for cross-reactivity between species. In addition, the newly developed mAbs were compared to mAbs used in previous research for their epitope recognition and other Sn-specific characteristics. Both species-specific and cross-reactive antibodies could be identified. Furthermore, sialic acid-binding of red blood cells (RBC) could be inhibited with mAbs recognizing different epitopes and all mAb showed internalization of Sn. The newly developed mAbs can be used as novel tools for Sn research and further analysis of Sn internalization in different species.