Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Fungi (Basel) ; 9(11)2023 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-37998902

RESUMO

The endocytic and secretory pathways of the fungal pathogen Candida albicans are fundamental to various key cellular processes such as cell growth, cell wall integrity, protein secretion, hyphal formation, and pathogenesis. Our previous studies focused on several candidate genes involved in early endocytosis, including ENT2 and END3, that play crucial roles in such processes. However, much remains to be discovered about other endocytosis-related genes and their contributions toward Candida albicans secretion and virulence. In this study, we examined the functions of the early endocytosis gene PAL1 using a reverse genetics approach based on CRISPR-Cas9-mediated gene deletion. Saccharomyces cerevisiae Pal1 is a protein in the early coat complex involved in clathrin-mediated endocytosis that is later internalized with the coat. The C. albicans pal1Δ/Δ null mutant demonstrated increased resistance to the antifungal agent caspofungin and the cell wall stressor Congo Red. In contrast, the null mutant was more sensitive to the antifungal drug fluconazole and low concentrations of SDS than the wild type (WT) and the re-integrant (KI). While pal1Δ/Δ can form hyphae and a biofilm, under some hyphal-inducing conditions, it was less able to demonstrate filamentous growth when compared to the WT and KI. The pal1Δ/Δ null mutant had no defect in clathrin-mediated endocytosis, and there were no changes in virulence-related processes compared to controls. Our results suggest that PAL1 has a role in susceptibility to antifungal agents, cell wall integrity, and membrane stability related to early endocytosis.

2.
Microbiol Spectr ; 11(3): e0536122, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37222596

RESUMO

While endocytic and secretory pathways are well-studied cellular processes in the model yeast Saccharomyces cerevisiae, they remain understudied in the opportunistic fungal pathogen Candida albicans. We previously found that null mutants of C. albicans homologs of the S. cerevisiae early endocytosis genes ENT2 and END3 not only exhibited delayed endocytosis but also had defects in cell wall integrity, filamentation, biofilm formation, extracellular protease activity, and tissue invasion in an in vitro model. In this study, we focused on a potential C. albicans homolog to S. cerevisiae TCA17, which was discovered in our whole-genome bioinformatics approach aimed at identifying genes involved in endocytosis. In S. cerevisiae, TCA17 encodes a transport protein particle (TRAPP) complex-associated protein. Using a reverse genetics approach with CRISPR-Cas9-mediated gene deletion, we analyzed the function of the TCA17 homolog in C. albicans. Although the C. albicans tca17Δ/Δ null mutant did not have defects in endocytosis, it displayed an enlarged cell and vacuole morphology, impaired filamentation, and reduced biofilm formation. Moreover, the mutant exhibited altered sensitivity to cell wall stressors and antifungal agents. When assayed using an in vitro keratinocyte infection model, virulence properties were also diminished. Our findings indicate that C. albicans TCA17 may be involved in secretion-related vesicle transport and plays a role in cell wall and vacuolar integrity, hyphal and biofilm formation, and virulence. IMPORTANCE The fungal pathogen Candida albicans causes serious opportunistic infections in immunocompromised patients and has become a major cause of hospital-acquired bloodstream infections, catheter-associated infections, and invasive disease. However, due to a limited understanding of Candida molecular pathogenesis, clinical approaches for the prevention, diagnosis, and treatment of invasive candidiasis need significant improvement. In this study, we focus on identifying and characterizing a gene potentially involved in the C. albicans secretory pathway, as intracellular transport is critical for C. albicans virulence. We specifically investigated the role of this gene in filamentation, biofilm formation, and tissue invasion. Ultimately, these findings advance our current understanding of C. albicans biology and may have implications for the diagnosis and treatment of candidiasis.


Assuntos
Candida albicans , Proteínas Fúngicas , Humanos , Candida albicans/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Parede Celular/metabolismo , Biofilmes , Hifas/metabolismo
3.
ACS Synth Biol ; 12(1): 153-163, 2023 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-36623275

RESUMO

Botulinum neurotoxin serotype A (BoNT/A) is a widely used cosmetic agent that also has diverse therapeutic applications; however, adverse antidrug immune responses and associated loss of efficacy have been reported in clinical uses. Here, we describe computational design and ultrahigh-throughput screening of a massive BoNT/A light-chain (BoNT/A-LC) library optimized for reduced T cell epitope content and thereby dampened immunogenicity. We developed a functional assay based on bacterial co-expression of BoNT/A-LC library members with a Förster resonance energy transfer (FRET) sensor for BoNT/A-LC enzymatic activity, and we employed high-speed fluorescence-activated cell sorting (FACS) to identify numerous computationally designed variants having wild-type-like enzyme kinetics. Many of these variants exhibited decreased immunogenicity in humanized HLA transgenic mice and manifested in vivo paralytic activity when incorporated into full-length toxin. One variant achieved near-wild-type paralytic potency and a 300% reduction in antidrug antibody response in vivo. Thus, we have achieved a striking level of BoNT/A-LC functional deimmunization by combining computational library design and ultrahigh-throughput screening. This strategy holds promise for deimmunizing other biologics with complex superstructures and mechanisms of action.


Assuntos
Anticorpos , Camundongos , Animais , Camundongos Transgênicos , Biblioteca Gênica , Domínios Proteicos
4.
Microbiol Spectr ; 10(2): e0188021, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35234488

RESUMO

The role of endocytosis in Candida albicans secretion, filamentation, and virulence remains poorly understood, despite its importance as a fundamental component of intracellular trafficking. Given that secretory mutants display defects in endocytosis, we have focused our attention on endocytic mutants to understand the interconnection between endocytosis and other secretory pathways. Using a reverse-genetic approach based upon CRISPR-Cas9 mediated gene deletion, we studied the functions of the gene END3, which plays a key role in clathrin-based endocytosis. In the end3Δ/Δ null mutant, clathrin-mediated endocytosis was substantially reduced. While in vitro growth, cell morphology, and vacuoles appeared normal, the mutant was impaired in actin patch formation, filamentous growth, biofilm formation, cell wall integrity, and extracellular protease secretion. In addition, susceptibility to various antifungal agents was altered. Consistent with the inability to form hyphae, in an in vitro keratinocyte infection model, the null mutant displayed reduced damage of mammalian adhesion zippers and host cell death. Thus, C. albicans END3 has a role in efficient endocytosis that is required for cell wall integrity, protein secretion, hyphal formation, and virulence-related processes. These findings suggest that impaired endocytosis subsequently affects other secretory pathways, providing evidence of the interconnection between these processes. IMPORTANCE Candida albicans is a fungal commensal organism that can cause serious opportunistic infections in immunocompromised patients leading to substantial complications and mortality. A better understanding of the microbe's biology to develop more effective therapeutic and diagnostic tools is required as invasive candidiasis is a problem of continued clinical importance. This study focuses on endocytosis, an important but incompletely understood cellular mechanism needed to uptake nutrients and communicate with a cell's environment. In this study, we have assessed the role of endocytosis in cell wall integrity, biofilm formation, and tissue invasion in C. albicans. These findings will improve our understanding of cellular mechanisms underlying endocytosis and will inform us of the interconnection with other intracellular transport processes.


Assuntos
Candida albicans , Proteínas Fúngicas , Animais , Parede Celular/metabolismo , Clatrina/metabolismo , Endocitose , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Hifas , Mamíferos/metabolismo
5.
mSphere ; 6(5): e0070721, 2021 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-34585966

RESUMO

Epsins play a pivotal role in the formation of endocytic vesicles and potentially provide a linkage between endocytic and other trafficking pathways. We identified a Candida albicans epsin, ENT2, that bears homology to the Saccharomyces cerevisiae early endocytosis genes ENT1 and ENT2 and studied its functions by a reverse genetic approach utilizing CRISPR-Cas9-mediated gene deletion. The C. albicans ent2Δ/Δ null mutant displayed cell wall defects and altered antifungal drug sensitivity. To define the role of C. albicans ENT2 in endocytosis, we performed assays with the lipophilic dye FM4-64 that revealed greatly reduced uptake in the ent2Δ/Δ mutant. Next, we showed that the C. albicans ent2Δ/Δ mutant was unable to form hyphae and biofilms. Assays for virulence properties in an in vitro keratinocyte infection model demonstrated reduced damage of mammalian adhesion zippers and host cell death from the ent2Δ/Δ mutant. We conclude that C. albicans ENT2 has a role in efficient endocytosis, a process that is required for maintaining cell wall integrity, hyphal formation, and virulence-defining traits. IMPORTANCE The opportunistic fungal pathogen Candida albicans is an important cause of invasive infections in hospitalized patients and a source of considerable morbidity and mortality. Despite its clinical importance, we still need to improve our ability to diagnose and treat this common pathogen. In order to support these advancements, a greater understanding of the biology of C. albicans is needed. In these studies, we are focused on the fundamental biological process of endocytosis, of which little is directly known in C. albicans. In addition to studying the function of a key gene in this process, we are examining the role of endocytosis in the virulence-related processes of filamentation, biofilm formation, and tissue invasion. These studies will provide greater insight into the role of endocytosis in causing invasive fungal infections.


Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/fisiologia , Parede Celular/microbiologia , Proteínas Fúngicas/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Antifúngicos/farmacologia , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Candida albicans/patogenicidade , Candidíase/microbiologia , Parede Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Hifas/citologia , Hifas/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Virulência
6.
Artigo em Inglês | MEDLINE | ID: mdl-33468459

RESUMO

Drug-resistant bacterial pathogens are a serious threat to global health, and antibacterial lysins are at the forefront of innovative treatments for these life-threatening infections. While lysins' general mechanism of action is well understood, the design principles that might enable engineering of performance-enhanced variants are still being formulated. Here, we report a detailed analysis of molecular determinants underlying the in vivo efficacy of lysostaphin, a canonical anti-MRSA (methicillin-resistant Staphylococcus aureus) lysin. Systematic analysis of bacterial binding, growth inhibition, lysis kinetics, and in vivo therapeutic efficacy revealed that binding affinity, and not inherent catalytic firepower, is the dominant driver of lysostaphin efficacy. This insight enabled electrostatic affinity tuning of lysostaphin to produce a single point mutant that manifested dramatically enhanced processivity and lysis kinetics and trended toward improved in vivo efficacy. More generally, these studies provide important insights into the complex relationships between lysin electrostatics, bacterial targeting, cell lysis efficiency, and in vivo efficacy. The lessons learned may enable engineering of other high-performance antibacterial biocatalysts.


Assuntos
Lisostafina , Staphylococcus aureus Resistente à Meticilina , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Cinética , Lisostafina/metabolismo , Lisostafina/farmacologia , Staphylococcus aureus Resistente à Meticilina/metabolismo , Eletricidade Estática
7.
Artigo em Inglês | MEDLINE | ID: mdl-33318001

RESUMO

There is an urgent need for novel agents to treat drug-resistant bacterial infections, such as multidrug-resistant Staphylococcus aureus (MRSA). Desirable properties for new antibiotics include high potency, narrow species selectivity, low propensity to elicit new resistance phenotypes, and synergy with standard-of-care (SOC) chemotherapies. Here, we describe analysis of the antibacterial potential exhibited by F12, an innovative anti-MRSA lysin that has been genetically engineered to evade detrimental antidrug immune responses in human patients. F12 possesses high potency and rapid onset of action, it has narrow selectivity against pathogenic staphylococci, and it manifests synergy with numerous SOC antibiotics. Additionally, resistance to F12 and ß-lactam antibiotics appears mutually exclusive, and, importantly, we provide evidence that F12 resensitizes normally resistant MRSA strains to ß-lactams both in vitro and in vivo These results suggest that combinations of F12 and SOC antibiotics are a promising new approach to treating refractory S. aureus infections.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Sinergismo Farmacológico , Humanos , Lisostafina/farmacologia , Testes de Sensibilidade Microbiana , Staphylococcus aureus , beta-Lactamas/farmacologia
8.
Sci Adv ; 6(36)2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32917596

RESUMO

There is a critical need for novel therapies to treat methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant pathogens, and lysins are among the vanguard of innovative antibiotics under development. Unfortunately, lysins' own microbial origins can elicit detrimental antidrug antibodies (ADAs) that undermine efficacy and threaten patient safety. To create an enhanced anti-MRSA lysin, a novel variant of lysostaphin was engineered by T cell epitope deletion. This "deimmunized" lysostaphin dampened human T cell activation, mitigated ADA responses in human HLA transgenic mice, and enabled safe and efficacious repeated dosing during a 6-week longitudinal infection study. Furthermore, the deimmunized lysostaphin evaded established anti-wild-type immunity, thereby providing significant anti-MRSA protection for animals that were immune experienced to the wild-type enzyme. Last, the enzyme synergized with daptomycin to clear a stringent model of MRSA endocarditis. By mitigating T cell-driven antidrug immunity, deimmunized lysostaphin may enable safe, repeated dosing to treat refractory MRSA infections.


Assuntos
Lisostafina , Staphylococcus aureus Resistente à Meticilina , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Lisostafina/farmacologia , Lisostafina/uso terapêutico , Camundongos , Camundongos Transgênicos
9.
AIDS Res Hum Retroviruses ; 36(10): 862-874, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32640832

RESUMO

Gene editing approaches using CRISPR/Cas9 are being developed as a means for targeting the integrated HIV-1 provirus. Enthusiasm for the use of gene editing as an anti-HIV-1 therapeutic has been tempered by concerns about the specificity and efficacy of this approach. Guide RNAs (gRNAs) that target conserved sequences across a wide range of genetically diverse HIV-1 isolates will have greater clinical utility. However, on-target efficacy should be considered in the context of off-target cleavage events as these may comprise an essential safety parameter for CRISPR-based therapeutics. We analyzed a panel of Streptococcus pyogenes Cas9 (SpCas9) gRNAs directed to the 5' and 3' long terminal repeat (LTR) regions of HIV-1. We used in vitro cleavage assays with genetically diverse HIV-1 LTR sequences to determine gRNA activity across HIV-1 clades. Lipid-based transfection of gRNA/Cas9 ribonucleoproteins was used to assess targeting of the integrated HIV-1 proviral sequence in cells (in vivo). For both the in vitro and in vivo experiments, we observed increased efficiency of sequence disruption through the simultaneous use of two distinct gRNAs. Next, CIRCLE-Seq was utilized to identify off-target cleavage events using genomic DNA from cells with integrated HIV-1 proviral DNA. We identified a gRNA targeting the U3 region of the LTR (termed SpCas9-127HBX2) with broad cleavage efficiency against sequences from genetically diverse HIV-1 strains. Based on these results, we propose a workflow for identification and development of anti-HIV CRISPR therapeutics.


Assuntos
Infecções por HIV , HIV-1 , Sistemas CRISPR-Cas , Edição de Genes , Infecções por HIV/genética , HIV-1/genética , Humanos , RNA Guia de Cinetoplastídeos/genética
10.
Vaccine ; 38(18): 3436-3446, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32192810

RESUMO

Antibodies against the HIV-1 V1V2 loops were the only correlate of reduced infection risk in the RV144 vaccine trial, highlighting the V1V2 loops as promising targets for vaccine design. The V1V2 loops are structurally plastic, exhibiting either an α-helix-coil or ß-strand conformation. V1V2-specific antibodies may thus recognize distinct conformations, and an antibody's conformational specificity can be an important determinant of breadth and function. Restricting V1V2 conformational plasticity in an immunogen may thus provide control over the conformational specificity and quality of a vaccine-elicited antibody response. Previously, we identified a V1V2 sequence variant (K155M) that results in enhanced recognition by cross-reactive antibodies recognizing the ß-strand conformation. Here, we relate V1V2 antigenicity to immunogenicity by comparing the immunogenicity profiles of wildtype and K155M immunogens in two mouse models. In one model, immunization with gp70 V1V2 K155M but not wildtype elicited antibody responses that were cross-reactive to a panel of heterologous gp120 and gp140 antigens. In a second model, we compared the effect of K155M on immunogenicity in the context of gp70 V1V2, gD V1V2 and gp120, examining the effects of scaffold, epitope-focusing and immunization regimen. K155M variants, especially in the context of a gp120 immunogen, resulted in more robust, durable and cross-reactive antibody responses than wildtype immunogens. Restriction of the ß-stranded V1V2 conformation in K155M immunogens may thus be associated with the induction of cross-reactive antibody responses thought to be required of a protective HIV-1 vaccine.


Assuntos
Vacinas contra a AIDS , Anticorpos Anti-HIV , Infecções por HIV , Animais , Anticorpos Neutralizantes , Formação de Anticorpos , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Camundongos
11.
J Fungi (Basel) ; 6(1)2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32102426

RESUMO

Candida albicans is a fungus that is a commensal organism and a member of the normal human microbiota. It has the ability to transition into an opportunistic invasive pathogen. Attributes that support pathogenesis include secretion of virulence-associated proteins, hyphal formation, and biofilm formation. These processes are supported by secretion, as defined in the broad context of membrane trafficking. In this review, we examine the role of secretory pathways in Candida virulence, with a focus on the model opportunistic fungal pathogen, Candida albicans.

12.
Math Med Biol ; 37(2): 212-242, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31265056

RESUMO

The use of gene-editing technology has the potential to excise the CCR5 gene from haematopoietic progenitor cells, rendering their differentiated CD4-positive (CD4+) T cell descendants HIV resistant. In this manuscript, we describe the development of a mathematical model to mimic the therapeutic potential of gene editing of haematopoietic progenitor cells to produce a class of HIV-resistant CD4+ T cells. We define the requirements for the permanent suppression of viral infection using gene editing as a novel therapeutic approach. We develop non-linear ordinary differential equation models to replicate HIV production in an infected host, incorporating the most appropriate aspects found in the many existing clinical models of HIV infection, and extend this model to include compartments representing HIV-resistant immune cells. Through an analysis of model equilibria and stability and computation of $R_0$ for both treated and untreated infections, we show that the proposed therapy has the potential to suppress HIV infection indefinitely and return CD4+ T cell counts to normal levels. A computational study for this treatment shows the potential for a successful 'functional cure' of HIV. A sensitivity analysis illustrates the consistency of numerical results with theoretical results and highlights the parameters requiring better biological justification. Simulations of varying level production of HIV-resistant CD4+ T cells and varying immune enhancements as the result of these indicate a clear threshold response of the model and a range of treatment parameters resulting in a return to normal CD4+ T cell counts.


Assuntos
Infecções por HIV/terapia , HIV-1 , Modelos Biológicos , Número Básico de Reprodução/estatística & dados numéricos , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Sistemas CRISPR-Cas , Biologia Computacional , Simulação por Computador , Edição de Genes/métodos , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Transplante de Células-Tronco Hematopoéticas/métodos , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Conceitos Matemáticos , Modelos Imunológicos , Receptores CCR5/deficiência , Receptores CCR5/genética
13.
Arthritis Res Ther ; 19(1): 270, 2017 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-29216931

RESUMO

BACKGROUND: In addition to activated T cells, the immune checkpoint inhibitor "V domain-containing Ig suppressor of T-cell activation" (VISTA) is expressed by myeloid cell types, including macrophages and neutrophils. The importance of VISTA expression by myeloid cells to antibody-induced arthritis and its potential for relevance in human disease was evaluated. METHODS: VISTA was immunolocalized in normal and arthritic human synovial tissue sections and synovial tissue lysates were subjected to western blot analysis. The collagen antibody-induced arthritis model (CAIA) was performed with DBA/1 J mice treated with antibodies against VISTA and with VISTA-deficient mice (V-KO). Total mRNA from arthritic joints, spleens, and cultured macrophages was analyzed with NanoString arrays. Cytokines secreted by splenic inflammatory macrophages were determined. In-vitro chemotaxis and signal transduction assays were performed with cultured macrophages. RESULTS: VISTA protein was localized to synovial membrane cells, neutrophils, and scattered cells in lymphocyte-rich foci and was detected by western blot analysis in normal synovium and synovium from rheumatoid arthritis patients. Deficiency of VISTA or treatment of mice with anti-VISTA monoclonal antibodies attenuated CAIA. Joint damage and MMP-3 expression were significantly reduced in V-KO mice. Surface expression of C5a receptor was reduced on monocytes, neutrophils, and cultured macrophages from V-KO. Upon Fc receptor engagement in vitro, gene expression by V-KO macrophages was altered profoundly compared to WT, including a significant induction of IL-1 receptor antagonist (IL1rn). CONCLUSIONS: VISTA expression supports immune-complex inflammation in CAIA and VISTA is expressed in human synovium. VISTA supports optimal responses to C5a and modulates macrophage responses to immune complexes.


Assuntos
Artrite Reumatoide/imunologia , Antígenos B7/imunologia , Regulação da Expressão Gênica/imunologia , Macrófagos/imunologia , Animais , Complexo Antígeno-Anticorpo/imunologia , Artrite Experimental/imunologia , Antígenos B7/deficiência , Humanos , Proteínas de Membrana/deficiência , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Membrana Sinovial/imunologia
14.
AIDS Res Hum Retroviruses ; 30(5): 457-67, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24328502

RESUMO

Innate immune responses to microbial pathogens are initiated following the binding of ligand to specific pattern recognition receptors. Each pattern recognition receptor, which includes members of the Toll-like receptor (TLR) family, is specific for a particular type of pathogen associated molecular pattern ensuring that the organism can respond rapidly to a wide range of pathogens including bacteria, viruses, and fungi. We studied the extent to which agonists to endosomal TLR could induce anti-HIV-1 activity in peripheral blood mononuclear cells (PBMCs). When agonists to TLR3, TLR7, TLR8 and TLR9 were added prior to infection with HIV-1, they significantly reduced infection of peripheral blood mononuclear cells. Interestingly, agonists to TLR8 and TLR9 were highly effective at blocking HIV replication even when added as late as 48 h or 72 h, respectively, after HIV-1 infection, indicating that the anti-viral effect was durable and long lasting. Analysis of the induction of anti-viral genes after agonist activation of TLR indicated that all of the agonists induced expression of the type I interferons and interferon stimulated genes, although to variable levels that depended on the agonist used. Interestingly, only the agonist to TLR9, ODN2395 DNA, induced expression of type II interferon and the anti-HIV proteins Apobec3G and SAMHD1. By blocking TLR activity using an inhibitor to the MyD88 adaptor protein, we demonstrated that, at least for TLR8 and TLR9, the anti-HIV activity was not entirely mediated by TLR activation, but likely by the activation of additional anti-viral sensors in HIV target cells. These findings suggest that agonists to the endosomal TLR function to induce expression of anti-HIV molecules by both TLR-mediated and non-TLR-mediated mechanisms. Moreover, the non-TLR-mediated mechanisms induced by these agonists could potentially be exploited to block HIV-1 replication in recently HIV-exposed individuals.


Assuntos
HIV-1/fisiologia , Imunidade Inata , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Receptores Toll-Like/agonistas , Replicação Viral , Adulto , Feminino , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
15.
AIDS Res Hum Retroviruses ; 29(6): 907-18, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23316755

RESUMO

Immune response modifiers are being studied as therapeutic agents for viral infections and cancer. These molecules include agonists for the Toll-like receptors (TLR), a family of innate immune receptors. TLR7 and 8, located in cellular endosomes, bind single-stranded RNA characteristic of viral genomes, and trigger intracellular signaling pathways that induce inflammatory cytokines and antiviral innate immune factors. We studied the anti-HIV-1 effects of gardiquimod, a specific TLR7 agonist when used at concentrations below 10 µM, in macrophages and activated peripheral blood mononuclear cells (PBMCs). Gardiquimod, added prior to or within 2 days after infection with X4, R5, or dual-tropic (R5/X4) strains of HIV-1, significantly reduced infection in these cells. Cocultures of activated PBMCs added to gardiquimod-treated and HIV-1-exposed macrophages demonstrated minimal HIV-1 replication for up to 10 days, suggesting that gardiquimod inhibited activated PBMCs viral amplification from HIV-1-exposed macrophages. Gardiquimod treatment of both activated PBMCs and macrophages induced interferon-alpha (IFN-α) transcription within hours of addition, and sustained IFN-α protein secretion for several days. Treatment of cells with a peptide inhibitor to the MyD88 adaptor protein blocked the induction of IFN-α by gardiquimod, and partially reversed the anti-HIV effects in activated PBMCs. Blocking the IFN-α receptor with a neutralizing antibody also reduced the anti-HIV effect of gardiquimod. Gardiquimod inhibited HIV-1 reverse transcriptase, an early step in the life cycle of HIV-1. These findings suggest that gardiquimod, functioning as both an immune system modifier and a reverse transcriptase inhibitor, could be developed as a novel therapeutic agent to block systemic and mucosal transmission of HIV-1.


Assuntos
Aminoquinolinas/uso terapêutico , Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Imidazóis/uso terapêutico , Macrófagos/virologia , Linfócitos T/virologia , Receptor 7 Toll-Like/agonistas , Linhagem Celular , DNA Viral/genética , HIV-1/fisiologia , Humanos , Interferon-alfa/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Macrófagos/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Linfócitos T/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
16.
Infect Dis Rep ; 3(2): e11, 2011 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-24470908

RESUMO

Human Immunodeficiency Virus-type 1 (HIV-1) binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA) transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV-1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA) sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α), a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.

17.
Open Immunol J ; 2: 86-93, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19966927

RESUMO

Cigarette smoke (CS) exposure is known to increase infection rates, but the mechanisms are not well understood. These studies tested the hypothesis that CS exposure would impair antimicrobial activity of apical conditioned media from human airway (BEAS-2B) cultures by reducing induction and release of the antimicrobial peptide CCL20. BEAS-2B cultures were exposed to CS extract and assayed for temporal and physical characteristics of release as well as for antimicrobial activity. E. coli were exposed to Beas-2B-conditioned media (BCM) and subsequent bacterial colonies were enumerated. In time course studies TLR-agonist-induced CCL20 transcription and release were rapid, of short duration and release was consistently targeted to the apical/luminal compartment. Cells treated with CS extract had diminished release of CCL20 under both constitutive and toll-like receptor (TLR) agonist stimulating conditions. Exposure of the cells to CS significantly reduced the antimicrobial activity in BCM and neutralizing antibodies to CCL20 brought antibacterial activity back to baseline levels demonstrating that antimicrobial activity in this culture system was primarily attributable to CCL20. These studies add to the understanding of CCL20 as a mucosal antimicrobial and improve insight into a likely mechanism linking infection to CS exposure.

18.
J Infect Dis ; 200(6): 965-72, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19671015

RESUMO

BACKGROUND: Mucosal surfaces of the female reproductive tract are the main routes of heterosexual transmission of human immunodeficiency virus type 1 (HIV-1), but the contribution of each of the reproductive sites to mucosal transmission is unknown. METHODS: We compared levels of HIV-1 transcription between ectocervical and endometrial tissue explants infected ex vivo with HIV-1. RESULTS: We detected higher levels of HIV-1 transcription in the ectocervix. Although CD45 expression was also increased at this site, higher levels of HIV-1 transcription could not be accounted for exclusively by differences in CD45 expression. This suggests that factors other than CD45 levels regulate HIV-1 transcription within the ectocervix. We detected higher levels of interleukin (IL)-6 at this site. Furthermore, addition of recombinant IL-6 to tissue explants enhanced HIV-1 transcription to a much greater degree in the ectocervix than in the endometrium. CONCLUSIONS: This is, to our knowledge, the first study to compare ectocervix and endometrium in a tissue explant model of HIV-1 infection and to demonstrate greater HIV-1 transcription in the ectocervix. Our results suggest that the ectocervix is more conducive to HIV-1 replication than is the endometrium and that IL-6 enhances HIV-1 transcription at this site. Thus, the ectocervix is an important site to be considered in heterosexual transmission of HIV-1.


Assuntos
Colo do Útero/virologia , Infecções por HIV/virologia , HIV-1 , Replicação Viral/fisiologia , Endométrio/virologia , Feminino , HIV-1/genética , Humanos , Interleucina-6/metabolismo , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Técnicas de Cultura de Tecidos , Transcrição Gênica
19.
J Acquir Immune Defic Syndr ; 51(2): 117-24, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19346967

RESUMO

BACKGROUND: Transmission of HIV from mother to child through breast-feeding remains a global health challenge, particularly in developing countries. Breast milk from an HIV-infected women may contain both cell-free HIV-1 and cell-associated virus; however, the impact of human breast milk on HIV infection and replication in CD4 cells remain poorly understood. OBJECTIVES: In the present study, we evaluated the effects of breast milk in vitro on infection of CD4 cells with cell-free HIV-1, including effects on HIV-1 receptor expression, reverse transcription, integration, and viral transcription. Additionally, we evaluated the ability of breast milk to inhibit cell-associated transmission of HIV-1 from infected CD4 T lymphocytes. RESULTS: Our results demonstrate that breast milk potently inhibits infection with cell-free HIV-1 in vitro independently of viral tropism and significantly decreases HIV-1 reverse transcription and integration in CD4 cells. However, the inhibitory effect of breast milk on HIV-1 infection of CD4 cells was lost during extended culture, and direct coculture of HIV-infected CD4 T lymphocytes with susceptible target cells revealed that breast milk was ineffective at blocking cell-associated HIV-1 infection. CONCLUSIONS: Our findings suggest that breast milk may provide a protective function against cell-free HIV-1 but may be less effective at blocking infection by cell-associated virus.


Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/efeitos dos fármacos , Leite Humano/química , Integração Viral/efeitos dos fármacos , Linhagem Celular , Sistema Livre de Células , Regulação Viral da Expressão Gênica/fisiologia , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/metabolismo , Humanos , Fatores de Tempo
20.
AIDS Res Hum Retroviruses ; 24(5): 701-16, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18462082

RESUMO

Endogenous levels of estradiol and progesterone fluctuate in the peripheral blood of premenopausal women during the reproductive cycle. We studied the effects of these sex hormones on HIV-1 replication in peripheral blood mononuclear cells (PBMCs). We compared HIV-1 replication in PBMCs infected in the presence of mid-secretory (high concentrations) and mid-proliferative (low concentrations) or in the absence of sex hormones. With PBMCs from men, we used concentrations of estradiol and progesterone that are normally present in their plasma. Our findings demonstrate that mid-proliferative phase conditions increased, and mid-secretory phase conditions decreased, HIV-1 replication. To determine if sex hormones affect specific stages of the viral life cycle we performed real-time PCR assays and found decreased levels of HIV-1 integration in the mid-secretory phase and increased levels viral transcription in the mid-proliferative phase. No significant effects on HIV-1 reverse transcription or on CCR5 expression were found. In addition, we assessed hormonal regulation of the HIV-1 LTR in the absence of the viral regulatory protein Tat. We observed that mid-proliferative hormone levels enhanced, whereas mid-secretory hormone concentrations reduced, the activity of the LTR. These findings demonstrate that in HIV-1-infected cells, estradiol and progesterone regulate HIV-1 replication most likely by directly altering HIV-1 transcriptional activation. An additional indirect mechanism of sex hormone regulation of cytokine and chemokine secretion cannot be excluded.


Assuntos
Estradiol/farmacologia , Infecções por HIV/virologia , HIV-1/fisiologia , Progesterona/farmacologia , Células Cultivadas , Feminino , Regulação Viral da Expressão Gênica , Repetição Terminal Longa de HIV/efeitos dos fármacos , Repetição Terminal Longa de HIV/fisiologia , Humanos , Leucócitos Mononucleares , Masculino , Transcrição Gênica/genética , Replicação Viral/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...