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1.
J Fungi (Basel) ; 7(3)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799778

RESUMO

Pleurotus ostreatus mushroom preparations have been investigated because of their ability to modulate the immune function. However, there is still no consensus regarding the activation and polarizing effect on macrophages by Pleurotus-derived bioproducts. This study examined the immune-activating effect of a mycelium-derived P. ostreatus aqueous extract (HW-Pm) on macrophage functions, by means of the determination of nitric oxide (NO) production, the mRNA expression of inducible nitric oxide synthase (iNOS), Arginase-1 and FIZZ and the cytokine levels. The phagocytic activity and the activation of NF-κB in U937 reporter cells were also investigated. No cytotoxicity was observed in macrophages treated with HW-Pm (IC50 > 1024 µg/mL) by the resazurin test. HW-Pm induced high levels of NO production and iNOS expression in macrophages. In contrast, HW-Pm did not induce Arginase-1 and FIZZ mRNA expressions. The mushroom extract increased TNF-α and IL-6 production and the phagocytic function in murine macrophages. It also stimulated the activation of the NF-κB promoter. The P. ostreatus mycelium extract has a potential application as a natural immune-enhancing agent, by targeting macrophage activation towards the classically activated subset and stimulating macrophage-mediated innate immune responses.

2.
Trends Mol Med ; 2021 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-33879402

RESUMO

Emerging evidence suggests that microbial therapeutics can prevent and treat respiratory viral diseases, especially when applied directly to the airways. This review presents established beneficial effects of locally administered microbial therapeutics against respiratory viral diseases and the inferred related molecular mechanisms. Several mechanisms established in the intestinal probiotics field as well as novel, niche-specific insights are relevant in the airways. Studies at cellular and organism levels highlight biologically plausible but strain-specific and host and virus context-dependent mechanisms, underlying the potential of beneficial bacteria. Large-scale clinical studies can now be rationally designed to provide a bench-to-bedside translation of the multifactorial bacterial mechanisms within the host respiratory tract, to diminish the incidence and severity of viral infections and the concomitant complications.

3.
Nanomedicine (Lond) ; 15(27): 2671-2688, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33112210

RESUMO

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.

4.
Sci Rep ; 10(1): 17268, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33057006

RESUMO

Dry eye syndrome (DES), a multifactorial disorder which leads to ocular discomfort, visual disturbance and tear film instability, has a rising prevalence and limited treatment options. In this study, a newly developed trypsin-like serine protease inhibitor (UAMC-00050) in a tear drop formulation was evaluated to treat ocular inflammation. A surgical animal model of dry eye was employed to investigate the potential of UAMC-00050 on dry eye pathology. Animals treated with UAMC-00050 displayed a significant reduction in ocular surface damage after evaluation with sodium fluorescein, compared to untreated, vehicle treated and cyclosporine-treated animals. The concentrations of IL-1α and TNF-α were also significantly reduced in tear fluid from UAMC-00050-treated rats. Additionally, inflammatory cell infiltration in the palpebral conjunctiva (CD3 and CD45), was substantially reduced. An accumulation of pro-MMP-9 and a decrease in active MMP-9 were found in tear fluid from animals treated with UAMC-00050, suggesting that trypsin-like serine proteases play a role in activating MMP-9 in ocular inflammation in this animal model. Comparative qRT-PCR analyses on ocular tissue indicated the upregulation of tryptase, urokinase plasminogen activator receptor (uPAR) and protease-activated receptor 2 (PAR2). The developed UAMC-00050 formulation was stable up to 6 months at room temperature in the absence of light, non-irritating and sterile with compatible pH and osmolarity. These results provide a proof-of-concept for the in vivo modifying potential of UAMC-00050 on dry eye pathology and suggest a central role of trypsin-like serine proteases and PAR2 in dry eye derived ocular inflammation.

5.
Front Microbiol ; 11: 1596, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760374

RESUMO

Pneumonia, of which Streptococcus pneumoniae is the most common causative agent, is considered one of the three top leading causes of death worldwide. As seen in other bacterial species, antimicrobial resistance is on the rise for this pathogen. Therefore, there is a pressing need for novel antimicrobial strategies to combat these infections. Recently, uridine diphosphate glucose pyrophosphorylase (UDPG:PP) has been put forward as a potential drug target worth investigating. Moreover, earlier research demonstrated that streptococci lacking a functional galU gene (encoding for UDPG:PP) were characterized by significantly reduced in vitro and in vivo virulence. Therefore, in this study we evaluated the anti-virulence activity of potential UDPG:PP inhibitors. They were selected in silico using a tailor-made streptococcal homology model, based on earlier listerial research. While the compounds didn't affect bacterial growth, nor affected in vitro adhesion to and phagocytosis in macrophages, the amount of polysaccharide capsule was significantly reduced after co-incubation with these inhibitors. Moreover, co-incubation proved to have a positive effect on survival in an in vivo Galleria mellonella larval infection model. Therefore, rather than targeting bacterial survival directly, these compounds proved to have an effect on streptococcal virulence by lowering the amount of polysaccharide and thereby probably boosting recognition of this pathogen by the innate immune system. While the compounds need adaptation to broaden their activity to more streptococcal strains rather than being strain-specific, this study consolidates UDPG:PP as a potential novel drug target.

6.
Front Immunol ; 11: 1113, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32582193

RESUMO

Type I interferons (IFNs) induced by an endogenous Leishmania RNA virus or exogenous viral infections have been shown to exacerbate infections with New World Cutaneous Leishmania parasites, however, the impact of type I IFNs in visceral Leishmania infections and implicated mechanisms remain to be unraveled. This study assessed the impact of type I IFN on macrophage infection with L. infantum and L. donovani and the implication of sialoadhesin (Siglec-1/CD169, Sn) as an IFN-inducible surface receptor. Stimulation of bone marrow-derived macrophages with type I IFN (IFN-α) significantly enhanced susceptibility to infection of reference laboratory strains and a set of recent clinical isolates. IFN-α particularly enhanced promastigote uptake. Enhanced macrophage susceptibility was linked to upregulated Sn surface expression as a major contributing factor to the infection exacerbating effect of IFN-α. Stimulation experiments in Sn-deficient macrophages, macrophage pretreatment with a monoclonal anti-Sn antibody or a novel bivalent anti-Sn nanobody and blocking of parasites with soluble Sn restored normal susceptibility levels. Infection of Sn-deficient mice with bioluminescent L. infantum promastigotes revealed a moderate, strain-dependent role for Sn during visceral infection under the used experimental conditions. These data indicate that IFN-responsive Sn expression can enhance the susceptibility of macrophages to infection with visceral Leishmania promastigotes and that targeting of Sn may have some protective effects in early infection.

7.
Viruses ; 11(11)2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31698728

RESUMO

Respiratory Syncytial Virus (RSV) is a very important viral pathogen in children, immunocompromised and cardiopulmonary diseased patients and the elderly. Most of the published research with RSV was performed on RSV Long and RSV A2, isolated in 1956 and 1961, yet recent RSV isolates differ from these prototype strains. Additionally, these viruses have been serially passaged in cell culture, which may result in adaptations that affect virus-host interactions. We have isolated RSV from mucosal secretions of 12 patients in the winters 2016-2017 and 2017-2018, of which eight RSV-A subtypes and four RSV-B subtypes. Passage 3 of the isolates was assessed for viral replication kinetics and infectious virus production in HEp-2, A549 and BEAS-2B cells, thermal stability at 37 °C, 32 °C and 4 °C, syncytia formation, neutralization by palivizumab and mucin mRNA expression in infected A549 cells. We observed that viruses isolated in one RSV season show differences on the tested assays. Furthermore, comparison with RSV A2 and RSV B1 reveals for some RSV isolates differences in viral replication kinetics, thermal stability and fusion capacity. Major differences are, however, not observed and differences between the recent isolates and reference strains is, overall, similar to the observed variation in between the recent isolates. One clinical isolate (BE/ANT-A11/17) replicated very efficiently in all cell lines, and remarkably, even better than RSV A2 in the HEp-2 cell line.


Assuntos
Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano/isolamento & purificação , Células A549 , Bélgica/epidemiologia , Bronquiolite/virologia , Linhagem Celular , Criança , Pré-Escolar , Humanos , Mucinas/metabolismo , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/virologia , Estações do Ano , Replicação Viral
8.
Intervirology ; 62(3-4): 134-144, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31533107

RESUMO

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.


Assuntos
Especificidade de Hospedeiro , Macrófagos/virologia , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Replicação Viral , Animais , Linhagem Celular , Humanos , Camundongos , Carga Viral , Ensaio de Placa Viral
9.
Eur J Med Chem ; 181: 111549, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31376569

RESUMO

Tuberculosis (TB) still has a major impact on public health. In order to efficiently eradicate this life-threatening disease, the exploration of novel anti-TB drugs is of paramount importance. As part of our program to design new 2-azaanthraquinones with anti-mycobacterial activity, various "out-of-plane" tetrahydro- and octahydrobenzo[j]phenanthridinediones were synthesized. In this study, the scaffold of the most promising hits was further optimized in an attempt to improve the bioactivity and to decrease enzymatic degradation. The rudiment bio-evaluation of a small library of fluorinated tetrahydrobenzo[j]phenanthridine-7,12-dione derivatives indicated no significant improvement of the bio-activity against intracellular and extracellular Mycobacterium tuberculosis (Mtb). Though, the derivatives showed an acceptable toxicity against J774A.1 macrophages and early signs of genotoxicity were absent. All derivatives showed to be metabolic stabile in the presence of both phase I and phase II murine or human microsomes. Finally, the onset of reactive oxygen species within Mtb after exposure to the derivatives was measured by electron paramagnetic resonance (EPR). Results showed that the most promising fluorinated derivative is still a possible candidate for the subversive inhibition of mycothione reductase.


Assuntos
Antituberculosos/farmacologia , Benzofenantridinas/farmacologia , Hidrocarbonetos Fluorados/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Animais , Antituberculosos/síntese química , Antituberculosos/química , Benzofenantridinas/síntese química , Benzofenantridinas/química , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/química , Macrófagos/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/crescimento & desenvolvimento , Relação Estrutura-Atividade
10.
Virus Res ; 266: 58-68, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31004621

RESUMO

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization worldwide each year and there is presently no licensed vaccine to prevent severe RSV infections. Two major RSV glycoproteins, attachment (G) and fusion (F) protein, regulate viral replication and both proteins contain potential glycosylation sites which are highly variable for the G protein and conserved for the F protein among virus isolates. The RSV F sequence possesses five N-glycosylation sites located in the F2 subunit (N27 and N70), the p27 peptide (N116 and N126) and the F1 subunit (N500). The importance of RSV F N-glycosylation in virus replication and immunogenicity is not yet fully understood, and a better understanding may provide new insights for vaccine development. By using a BAC-based reverse genetics system, recombinant viruses expressing F proteins with loss of N-glycosylation sites were made. Mutant viruses with single N-glycosylation sites removed could be recovered, while this was not possible with the mutant with all N-glycosylation sites removed. Although the individual RSV F N-glycosylation sites were shown not to be essential for viral replication, they do contribute to the efficiency of in vitro and in vivo viral infection. To evaluate the role of N-glycosylation sites on RSV F antigenicity, serum antibody titers were determined after infection of BALB/c mice with RSV expressing the glycomutant F proteins. Infection with recombinant virus lacking the N-glycosylation site at position N116 (RSV F N116Q) resulted in significant higher neutralizing antibody titers compared to RSV F WT infection, which is surprising since this N-glycan is present in the p27 peptide which is assumed to be absent from the mature F protein in virions. Thus, single or combined RSV F glycomutations which affect virus replication and fusogenicity, and which may induce enhanced antibody responses upon immunization could have the potential to improve the efficacy of RSV LAV approaches.


Assuntos
Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Vírus Sincicial Respiratório Humano/patogenicidade , Proteínas Virais de Fusão/metabolismo , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Feminino , Células Gigantes/virologia , Glicosilação , Humanos , Imunização , Imunogenicidade da Vacina , Camundongos Endogâmicos BALB C , Mutação , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/patologia , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Vírus Sincicial Respiratório Humano/imunologia , Células Vero , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Replicação Viral
11.
Sci Rep ; 9(1): 2900, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814593

RESUMO

Lactobacilli have been evaluated as probiotics against Candida infections in several clinical trials, but with variable results. Predicting and understanding the clinical efficacy of Lactobacillus strains is hampered by an overall lack of insights into their modes of action. In this study, we aimed to unravel molecular mechanisms underlying the inhibitory effects of lactobacilli on hyphal morphogenesis, which is a crucial step in C. albicans virulence. Based on a screening of different Lactobacillus strains, we found that the closely related taxa L. rhamnosus, L. casei and L. paracasei showed stronger activity against Candida hyphae formation compared to other Lactobacillus species tested. By exploring the activity of purified compounds and mutants of the model strain L. rhamnosus GG, the major peptidoglycan hydrolase Msp1, conserved in the three closely related taxa, was identified as a key effector molecule. We could show that this activity of Msp1 was due to its ability to break down chitin, the main polymer in the hyphal cell wall of C. albicans. This identification of a Lactobacillus-specific protein with chitinase activity having anti-hyphal activity will assist in better strain selection and improved application in future clinical trials for Lactobacillus-based Candida-management strategies.


Assuntos
Proteínas de Bactérias/metabolismo , Candida albicans/fisiologia , Candidíase/terapia , Quitinases/metabolismo , Hifas/fisiologia , Lactobacillus rhamnosus/fisiologia , Probióticos/uso terapêutico , Terapia Biológica , Candida albicans/patogenicidade , Quitina/metabolismo , Humanos , Morfogênese , Interferência de RNA , RNA Ribossômico 16S/genética , Especificidade da Espécie , Virulência
12.
Front Microbiol ; 10: 311, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30846978

RESUMO

Streptococcus pneumoniae is the leading cause of bacterial pneumonia. Infection is linked to high morbidity and mortality rates and antibiotic resistance within this pathogen is on the rise. Therefore, there is a need for novel antimicrobial therapies. To lower the time and costs of the drug discovery process, alternative in vivo models should be considered. As such, Galleria mellonella larvae can be of great value. The larval immunity consisting of several types of haemocytes is remarkably similar to the human innate immune system. Furthermore, these larvae don't require specific housing, are cheap and are easy to handle. In this study, the use of a G. mellonella infection model to study early pneumococcal infections and treatment is proposed. Firstly, the fitness of this model to study pneumococcal virulence factors is confirmed using streptococcal strains TIGR4, ATCC®49619, D39 and its capsule-deficient counterpart R6 at different inoculum sizes. The streptococcal polysaccharide capsule is considered the most important virulence factor without which streptococci are unable to sustain an in vivo infection. Kaplan-Meier survival curves showed indeed a higher larval survival after infection with streptococcal strain R6 compared to strain D39. Then, the infection was characterized by determining the number of haemocytes, production of oxygen free radicals and bacterial burden at several time points during the course of infection. Lastly, treatment of infected larvae with the standard antibiotics amoxicillin and moxifloxacin was evaluated. Treatment has proven to have a positive outcome on the course of infection, depending on the administered dosage. These data imply that G. mellonella larvae can be used to evaluate antimicrobial therapies against S. pneumoniae, apart from using the larval model to study streptococcal properties. The in-depth knowledge acquired regarding this model, makes it more suitable for use in future research.

13.
J Antimicrob Chemother ; 74(2): 395-406, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30412253

RESUMO

Objectives: Miltefosine is currently the only oral drug for visceral leishmaniasis, and although deficiency in an aminophospholipid/miltefosine transporter (MT) is sufficient to elicit drug resistance, very few naturally miltefosine-resistant (MIL-R) strains have yet been isolated. This study aimed to make a detailed analysis of the impact of acquired miltefosine resistance and miltefosine treatment on in vivo infection. Methods: Bioluminescent versions of a MIL-R strain and its syngeneic parental line were generated by integration of the red-shifted firefly luciferase PpyRE9. The fitness of both lines was compared in vitro (growth rate, metacyclogenesis and macrophage infectivity) and in BALB/c mice through non-invasive bioluminescence imaging under conditions with and without drug pressure. Results: This study demonstrated a severe fitness loss of MT-deficient parasites, resulting in a complete inability to multiply and cause a typical visceral leishmaniasis infection pattern in BALB/c mice. The observed fitness loss could not be rescued by host immune suppression with cyclophosphamide, whereas episomal reconstitution with a wild-type MT restored parasite virulence, hence linking parasite fitness to MT mutation. Remarkably, in vivo miltefosine treatment or in vitro miltefosine pre-exposure significantly rescued MIL-R parasite virulence. The in vitro pre-exposed MIL-R promastigotes showed a longer and more slender morphology, suggesting an altered membrane composition. Conclusions: The profound fitness loss of MT-deficient parasites most likely explains the low frequency of MIL-R clinical isolates. The observation that miltefosine can reverse this phenotype indicates a drug dependency of the MT-deficient parasites and emphasizes the importance of resistance profiling prior to miltefosine administration.


Assuntos
Aptidão Genética/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/genética , Macrófagos/parasitologia , Proteínas de Membrana Transportadoras/genética , Fosforilcolina/análogos & derivados , Animais , Feminino , Imunossupressão , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Visceral/parasitologia , Luciferases/metabolismo , Medições Luminescentes , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Testes de Sensibilidade Parasitária , Fosforilcolina/farmacologia , Virulência/efeitos dos fármacos
14.
Cancer Immunol Immunother ; 68(4): 673-685, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30569204

RESUMO

Many pathogens, ranging from viruses to multicellular parasites, promote expansion of MDSCs, which are myeloid cells that exhibit immunosuppressive features. The roles of MDSCs in infection depend on the class and virulence mechanisms of the pathogen, the stage of the disease, and the pathology associated with the infection. This work compiles evidence supported by functional assays on the roles of different subsets of MDSCs in acute and chronic infections, including pathogen-associated malignancies, and discusses strategies to modulate MDSC dynamics to benefit the host.


Assuntos
Doenças Transmissíveis/etiologia , Doenças Transmissíveis/metabolismo , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Doença Aguda , Animais , Biomarcadores , Doença Crônica , Doenças Transmissíveis/tratamento farmacológico , Suscetibilidade a Doenças , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunomodulação , Terapia de Alvo Molecular , Células Supressoras Mieloides/efeitos dos fármacos
15.
Viruses ; 10(8)2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30110893

RESUMO

Prevention of severe lower respiratory tract infections in infants caused by the human respiratory syncytial virus (hRSV) remains a major public health priority. Currently, the major focus of vaccine development relies on the RSV fusion (F) protein since it is the main target protein for neutralizing antibodies induced by natural infection. The protein conserves 5 N-glycosylation sites, two of which are located in the F2 subunit (N27 and N70), one in the F1 subunit (N500) and two in the p27 peptide (N116 and N126). To study the influence of the loss of one or more N-glycosylation sites on RSV F immunogenicity, BALB/c mice were immunized with plasmids encoding RSV F glycomutants. In comparison with F WT DNA immunized mice, higher neutralizing titres were observed following immunization with F N116Q. Moreover, RSV A2-K-line19F challenge of mice that had been immunized with mutant F N116Q DNA was associated with lower RSV RNA levels compared with those in challenged WT F DNA immunized animals. Since p27 is assumed to be post-translationally released after cleavage and thus not present on the mature RSV F protein, it remains to be elucidated how deletion of this glycan can contribute to enhanced antibody responses and protection upon challenge. These findings provide new insights to improve the immunogenicity of RSV F in potential vaccine candidates.


Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas de DNA/administração & dosagem , Proteínas Virais de Fusão/genética , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Feminino , Glicosilação , Humanos , Hidrólise , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação , Plasmídeos/administração & dosagem , Plasmídeos/genética , Plasmídeos/imunologia , Engenharia de Proteínas , Subunidades Proteicas/administração & dosagem , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/genética , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Proteínas Virais de Fusão/administração & dosagem , Proteínas Virais de Fusão/imunologia , Carga Viral/efeitos dos fármacos
16.
J Mol Diagn ; 20(2): 253-263, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29355825

RESUMO

Several methods have been developed for the detection of Leishmania, mostly targeting the minicircle kinetoplast DNA (kDNA). A new RNA real-time quantitative PCR (qPCR) assay was developed targeting the conserved and highly expressed spliced-leader (SL) mini-exon sequence. This study compared the limits of detection of various real-time PCR assays in hamsters infected with Leishmania infantum, in spiked human blood, and in clinical blood samples from visceral leishmaniasis patients. The SL-RNA assay showed an excellent analytical sensitivity in tissues (0.005 and 0.002 parasites per mg liver and spleen, respectively) and was not prone to false-positive reactions. Evaluation of the SL-RNA assay on clinical samples demonstrated lower threshold cycle values than the kDNA qPCR, an excellent interrun stability of 97%, a 93% agreement with the kDNA assay, and an estimated sensitivity, specificity, and accuracy of 93.2%, 94.3%, and 93.8%, respectively. The SL-RNA qPCR assay was equally efficient for detecting Leishmania major, Leishmania tropica, Leishmania mexicana, Leishmania guayensis, Leishmania panamensis, Leishmania braziliensis, L. infantum, and Leishmania donovani and revealed similar SL-RNA levels in the different species and the occurrence of polycistronic SL-containing transcripts in Viannia species. Collectively, this single SL-RNA qPCR assay enables universal Leishmania detection and represents a particularly useful addition to the widely used kDNA assay in clinical studies in which the detection of viable parasites is pivotal to assess parasitological cure.


Assuntos
DNA de Cinetoplasto/análise , Leishmania infantum/genética , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Mesocricetus/parasitologia , RNA Líder para Processamento/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Criança , Pré-Escolar , Cricetinae , Confiabilidade dos Dados , Feminino , Humanos , Fígado/parasitologia , Sensibilidade e Especificidade , Baço/parasitologia
17.
Mult Scler ; 24(3): 290-300, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28277099

RESUMO

BACKGROUND: Phagocytes, such as macrophages and microglia, are key effector cells in the pathophysiology of multiple sclerosis (MS). It is widely accepted that they instigate and promote neuroinflammatory and neurodegenerative events in MS. An increasing amount of studies indicate that Siglec-1, also known CD169, is a marker for activated phagocytes in inflammatory disorders. OBJECTIVE: In this study, we set out to define how CD169+ phagocytes contribute to neuroinflammation in MS. METHODS: CD169-diphtheria toxin receptor (DTR) mice, which express human DTR under control of the CD169 promoter, were used to define the impact of CD169+ cells on neuroinflammation. Flow cytometry and immunohistochemistry were utilized to determine the expression and distribution of CD169. RESULTS: We show that CD169 is highly expressed by lesional and circulating phagocytes in MS and experimental autoimmune encephalomyelitis (EAE). Our data further indicate that CD169 represents a selective marker for early activated microglia in MS and EAE lesions. Depletion of CD169+ cells markedly reduced neuroinflammation and ameliorated disease symptoms in EAE-affected mice. CONCLUSION: Our findings indicate that CD169+ cells promote neuroinflammation. Furthermore, they suggest that CD169+ phagocytes play a key role in the pathophysiology of MS. Hence, targeting CD169+ phagocytes may hold therapeutic value for MS.


Assuntos
Biomarcadores , Encefalomielite Autoimune Experimental/diagnóstico , Inflamação/diagnóstico , Esclerose Múltipla/diagnóstico , Fagócitos , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade
18.
Chem Biol Drug Des ; 91(2): 631-640, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28845550

RESUMO

The diverse pharmacological properties of the diaryltriazenes have sparked the interest to investigate their potential to be repurposed as antitubercular drug candidates. In an attempt to improve the antitubercular activity of a previously constructed diaryltriazene library, eight new halogenated nitroaromatic triazenides were synthesized and underwent biological evaluation. The potency of the series was confirmed against the Mycobacterium tuberculosis lab strain H37Ra, and for the most potent derivative, we observed a minimal inhibitory concentration of 0.85 µm. The potency of the triazenide derivatives against M. tuberculosis H37Ra was found to be highly dependent on the nature of the halogenated phenyl substituent and less dependent on cationic species used for the preparation of the salts. Although the inhibitory concentration against J774A.1 macrophages was observed at 3.08 µm, the cellular toxicity was not mediated by the generation of nitroxide intermediate as confirmed by electron paramagnetic resonance spectroscopy, whereas no in vitro mutagenicity could be observed for the new halogenated nitroaromatic triazenides when a trifluoromethyl substituent was present on both the aryl moieties.


Assuntos
Antituberculosos/química , Triazenos/química , Animais , Antituberculosos/síntese química , Antituberculosos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Espectroscopia de Ressonância de Spin Eletrônica , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Halogenação , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Nitrofenóis/química , Relação Estrutura-Atividade , Triazenos/síntese química , Triazenos/farmacologia
19.
Microb Biotechnol ; 10(6): 1753-1763, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28772020

RESUMO

A number of clinical studies have shown protective effects of lactobacilli against Candida species in the gastrointestinal tract, the urogenital tract and the oral cavity, while others did not show clear effects. Evidence on the mode of action of lactobacilli against Candida is also still lacking. In this study, the anti-Candida activity of the model probiotic strain Lactobacillus rhamnosus GG was explored in different assays to determine molecular interactions. We found that L. rhamnosus GG was able to interfere with Candida growth, morphogenesis and adhesion. These three aspects of Candida's physiology are all crucial to its opportunistic pathogenesis. In follow-up assays, we compared the activity of L. rhamnosus GG wild-type with its exopolysaccharide (EPS)-deficient mutant and purified EPS to evaluate the involvement of this outer carbohydrate layer. Our data demonstrate that purified EPS can both interfere with hyphal formation and adhesion to epithelial cells, which indicates that EPS is part of a combined molecular mechanism underlying the antihyphal and anti-adhesion mechanisms of L. rhamnosus GG.


Assuntos
Candida/efeitos dos fármacos , Candida/crescimento & desenvolvimento , Lactobacillus rhamnosus/metabolismo , Proteoglicanas/farmacologia , Candida/genética , Candida/fisiologia , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Lactobacillus rhamnosus/química , Lactobacillus rhamnosus/genética , Proteoglicanas/metabolismo
20.
Virol J ; 14(1): 157, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28818113

RESUMO

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.


Assuntos
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
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