RESUMEN
Ultrashort cationic lipopeptides (USCLs) and gemini cationic surfactants are classes of potent antimicrobials. Our recent study has shown that the branching and shortening of the fatty acids chains with the simultaneous addition of a hydrophobic N-terminal amino acid in USCLs result in compounds with enhanced selectivity. Here, this approach was introduced into arginine-rich gemini cationic surfactants. l-cystine diamide and l-lysine amide linkers were used as spacers. Antimicrobial activity against planktonic and biofilm cultures of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) strains and Candida sp. as well as hemolytic and cytotoxic activities were examined. Moreover, antimicrobial activity in the presence of human serum and the ability to form micelles were evaluated. Membrane permeabilization study, serum stability assay, and molecular dynamics were performed. Generally, critical aggregation concentration was linearly correlated with hydrophobicity. Gemini surfactants were more active than the parent USCLs, and they turned out to be selective antimicrobial agents with relatively low hemolytic and cytotoxic activities. Geminis with the l-cystine diamide spacer seem to be less cytotoxic than their l-lysine amide counterparts, but they exhibited lower antibiofilm and antimicrobial activities in serum. In some cases, geminis with branched fatty acid chains and N-terminal hydrophobic amino acid resides exhibited enhanced selectivity to pathogens over human cells.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/síntesis química , Biopelículas/efectos de los fármacos , Lipoproteínas/síntesis química , Tensoactivos/síntesis química , Secuencias de Aminoácidos , Péptidos Catiónicos Antimicrobianos/farmacología , Arginina/química , Candida/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Cistina/química , Enterobacteriaceae/efectos de los fármacos , Ácidos Grasos/química , Hemólisis , Interacciones Hidrofóbicas e Hidrofílicas , Lipoproteínas/farmacología , Lisina/química , Micelas , Tensoactivos/farmacologíaRESUMEN
Lipoproteins (LPs) are circulating heterogeneous nanoparticles produced by the liver and intestines. LPs play a major role in the transport of dietary and endogenous lipids to target cells through cell membrane receptors or cell surface-bound lipoprotein lipase. The stability, biocompatibility, and selective transport of LPs make them promising delivery vehicles for various therapeutic and imaging agents. This review discusses isolation, manufacturing, and drug loading techniques used for LP-based drug delivery, as well as recent applications for diagnosis and treatment of cancer, atherosclerosis, and other life-threatening diseases.
Asunto(s)
Sistemas de Liberación de Medicamentos , Lipoproteínas/administración & dosificación , Animales , Humanos , Lipoproteínas/biosíntesis , Lipoproteínas/síntesis química , Lipoproteínas/aislamiento & purificaciónRESUMEN
An efficient total synthesis of A54145 factor D (A5D), a member of the A54145 family of cyclic lipodepsipeptide antibiotics, is reported. The peptide was constructed by attaching the peptide to the 2'-chlorotrityl polystyrene resin via Sar5 and developing conditions that avoided diketopiperazine formation upon subsequent elaboration using 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. This route allowed for facile formation of the crucial depsi bond. A branched acyclic precursor was cyclized off-resin and then globally deprotected to obtain A5D. Consistent with recent studies by others, we found that the MeOAsp residue has the 2S,3R configuration. We also established that the configuration of the stereocenter in the anteiso-undecanoyl lipid tail does not affect biological activity.
Asunto(s)
Antibacterianos/síntesis química , Factor D del Complemento/síntesis química , Técnicas de Síntesis en Fase Sólida/métodos , Antibacterianos/química , Antibacterianos/farmacología , Factor D del Complemento/química , Factor D del Complemento/farmacología , Lipoproteínas/síntesis química , Lipoproteínas/química , Lipoproteínas/farmacología , Estructura Molecular , EstereoisomerismoRESUMEN
A54145 is a family of antibacterial cyclic lipodepsipeptides structurally resembling daptomycin. Since its discovery in 1990, only the ambiguous structures of the methoxy-aspartic acid (MeO-Asp) and the hydroxy-asparagine (HO-Asn) have been reported. We have developed efficient routes to obtain the fully protected l-MeO-Asp and l-HO-Asn building blocks compatible with Fmoc-SPPS, and a total synthesis of A54145 that enabled us to establish its structure, consisting of l-3S-HO-Asn and l-3R-MeO-Asp, revising the wrongly proposed structure of l-3S-MeO-Asp.
Asunto(s)
Antibacterianos/química , Antibacterianos/síntesis química , Técnicas de Química Sintética , Lipoproteínas/síntesis química , Lipoproteínas/química , Pruebas de Sensibilidad MicrobianaRESUMEN
The current study proposed that previously characterized individual antigenic proteins could represent potential replacement for conventional purified protein derivative (PPD) in tuberculosis skin testing when used in cocktails triggered by suitable TLR-stimulants that would provide the missing pro-inflammatory stimulus. Three different cocktails of previously selected antigens, including C1 (ESAT-6/CPF-10/MPB-83); C2 (ESAT-6/MPB-64/MPB-83); and C3 (CPF-10/MPB-64/MPB-83), were evaluated in vitro using lymphocytic proliferation and IFN-γ production assays, as well as mRNA and protein expression levels of TNF-α, IL-12p40, and IL-2 as pro-inflammatory molecules. C1 showed the highest significant induction of pro-inflammatory molecules as compared to other cocktails, yet still significantly lower than that induced by conventional PPD. Interestingly, inclusion of the synthetic Mycobacterium tuberculosis 19-kDa lipoprotein (Pam3Cys-SSNKSTTGSGETTTA) as a TLR-stimulant resulted in obvious augmentation of C1-induced pro-inflammatory molecules to levels comparable to that of PPD. In addition, skin testing using sensitized guinea pig model revealed comparable significant reaction to that of conventional PPD. ESAT-6/CPF-10/MPB-83 cocktail is suggested as a potential alternative skin-testing reagent when used in combination with the M. tuberculosis 19-kDa lipoprotein as a TLR-stimulant.
Asunto(s)
Antígenos Bacterianos/inmunología , Hipersensibilidad Tardía/inmunología , Lipoproteínas/inmunología , Mycobacterium tuberculosis/inmunología , Receptores Toll-Like/inmunología , Animales , Cobayas , Lipoproteínas/síntesis química , Lipoproteínas/química , Prueba de TuberculinaRESUMEN
Amphiphysin 2 and members of the BAR-domain family of proteins participate in a wide array of cellular processes including cell cycle and endocytosis. Given that amphiphysin 2 is related to diverse cell responses as a result of metabolic stress, we investigated in macrophages whether oxidative stress originated by the internalization of oxidized low density lipoproteins (oxLDL) affect both, the expression of amphiphysin 2 and its binding partner c-Myc. Here we report that under oxidative stress, a complex formation between amphiphysin 2(Bin1) and c-Myc allows the cell to develop a novel survival equilibrium state established between cell proliferation and cell death. We propose that under conditions of oxidative stress given by the internalization of oxLDL, macrophages employ the formation of the amphiphysin 2(Bin1)/c-Myc complex as a control mechanism to initially avoid the process of cell death in an attempt to prolong cell survival.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Supervivencia Celular , Endocitosis , Lipoproteínas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Supervivencia Celular/fisiología , Células Cultivadas , Humanos , Lipoproteínas/síntesis química , Lipoproteínas LDL/metabolismo , Sustancias Macromoleculares/química , Macrófagos/citología , Macrófagos/metabolismo , Estrés OxidativoRESUMEN
Background: Several tools have been proposed for serodiagnosis of cystic echinococcosis (CE), but none seems promising for cyst viability assessment. Antigens with stage-specific diagnostic value have been described, but few studies with well-characterized antigens and human serum samples have been performed. Antigen B (AgB) proteoforms hold promise as markers of viability, due to their differential stage-related expression and immunoreactivity. Methods: Four AgB subunits (AgB1, AgB2, AgB3, AgB4) were synthesized and structurally characterized. Based on the preliminary evaluation of the subunits by western immunoblotting and enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results: All subunits showed a high degree of spontaneous oligomerization. Interacting residues within oligomers were identified, showing that both the N-terminal and C-terminal of each subunit are involved in homo-oligomer contact interfaces. No hetero-oligomer was identified. AgB1 and AgB2 ELISAs revealed different sensitivities relative to cyst stage. Of note, besides high specificity (97.2%), AgB1 revealed a higher sensitivity for active-transitional cysts (100% for CE1, 77.8% for CE2, 81.5% for CE3a, and 86.3% for CE3b) than for inactive cysts (41.7% for CE4 and 11.1% for CE5) and postsurgical patients (44%). Interestingly, 19 of 20 patients with spontaneously inactive cysts and 6 of 9 treated with albendazole >5 years earlier were negative on the AgB1 assay. Conclusions: The structural characterization of subunits provides insights into the synthetic antigen conformation. The stage-related sensitivity of synthetic AgB1 holds promise as part of a multiantigen setting and deserves further longitudinal evaluation as marker of cyst viability.
Asunto(s)
Equinococosis/diagnóstico , Echinococcus granulosus/inmunología , Proteínas del Helminto/química , Proteínas del Helminto/inmunología , Lipoproteínas/química , Lipoproteínas/inmunología , Secuencia de Aminoácidos , Animales , Equinococosis/inmunología , Ensayo de Inmunoadsorción Enzimática , Proteínas del Helminto/síntesis química , Humanos , Lipoproteínas/síntesis química , Sensibilidad y Especificidad , Pruebas SerológicasRESUMEN
A multicomponent macrocyclization strategy towards cyclic lipopeptides is described. The approach relies on the utilization of the Ugi and Passerini multicomponent reactions for the cyclization of peptides and oxo-peptides, and here it is employed for the construction of a small library of analogues of the natural products mycosubtilin and surfactin A. A key feature of this method is the simultaneous incorporation of either one or two exocyclic lipid tails along with the macrocyclic ring closure, which is only possible due to the multicomponent nature of the macrocyclization step. The evaluation of the anticancer activity of the lipopeptide library showed that the installation of a second lipid moiety in the surfactin scaffold leads to a more potent cytotoxicity in cancer cells. This is a new example of the multicomponent reaction potential in rapidly producing natural product analogues for biological screening.
Asunto(s)
Productos Biológicos/síntesis química , Productos Biológicos/farmacología , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Productos Biológicos/química , Línea Celular Tumoral , Técnicas de Química Sintética , Ciclización , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Lipoproteínas/síntesis química , Lipoproteínas/química , Lipoproteínas/farmacología , Péptidos Cíclicos/químicaRESUMEN
Synthetic lipoprotein is an effective carrier of targeted delivery for drugs. It has the very small size, good biocompatibility, suitable half-life, and specific lipoprotein receptorbinding capacity. Compared with the traditional natural lipoprotein, synthetic lipoprotein not only retains the original biological characteristics and functions, but also exhibits the excellent characteristics in drug delivery. Herein, the advantages, development, applications, and prospect of synthetic lipoproteins as drug carriers were summarized.
Asunto(s)
Portadores de Fármacos , Lipoproteínas , Animales , Portadores de Fármacos/síntesis química , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Descubrimiento de Drogas , Humanos , Lipoproteínas/síntesis química , Lipoproteínas/química , Lipoproteínas/metabolismo , Nanopartículas/química , Receptores de LDL/metabolismo , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Chirally pure R- and S-epimers of TLR2 ligand Pam3CysSK4 were prepared and separately conjugated to an OVA model epitope, in which lysine was replaced by azidonorleucine. The azide function in the conjugate permitted labelling with different fluorophores by use of strain-promoted 3+2 cycloaddition. The R-epimer of the labelled conjugates induced TLR2-dependent DC maturation, while S-epimer proved to be inactive. Combining the lipophilicity of Pam3CysSK4 ligand with fluorophores influenced the solubility of the resulting conjugates in an unpredictable way and only the conjugates labelled with Cy-5 were suitable for confocal fluorescence microscopy experiments. It was shown that both epimers of the Cy-5 labelled lipopeptides were internalized equally well, indicating TLR2-independent cellular uptake. The presented results demonstrate the usefulness of strain-promoted azide-alkyne cycloaddition in the labelling of highly lipophilic lipopeptides without disturbing the in vitro activity of these conjugates with respect to activation of TLR-2.
Asunto(s)
Cisteína/análogos & derivados , Colorantes Fluorescentes/síntesis química , Lipoproteínas/síntesis química , Línea Celular , Cisteína/síntesis química , Cisteína/química , Cisteína/farmacocinética , Células Dendríticas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacocinética , Humanos , Interleucina-8/biosíntesis , Ligandos , Lipoproteínas/química , Lipoproteínas/farmacocinética , Estructura Molecular , Solubilidad , Relación Estructura-Actividad , Receptor Toll-Like 2/metabolismoRESUMEN
Neutrophil granulocytes are paramount to innate responses as major effectors of acute inflammation. Among the various strategies enacted by neutrophils to eliminate microbes NETosis is a novel distinct antimicrobial activity in which an interlacement of chromatin fibres rich in granule-derived antimicrobial peptides and enzymes is extruded (NETs, neutrophils extracellular traps ). NETs contribute to the pathogenesis of acute and chronic inflammatory disorders. The interactions of mycoplasmas and innate immune cells, in particular neutrophil granulocytes, are poorly defined. Here, we describe NET formation in vivo in the mammary gland and milk of sheep naturally infected by Mycoplasma agalactiae. Also, we assess the contribution of liposoluble proteins, the most abundant component of the Mycoplasma membrane, in inducing NETosis. We demonstrate that Mycoplasma liposoluble proteins induce NET release at levels comparable to what observed with other stimuli, such as lipopolysaccharides and phorbol 12-myristate 13-acetate. Stimulation of neutrophils with synthetic diacylated lipopeptides based on the M. agalactiae P48, P80, and MAG_1000 proteins, combined in a mix or used individually, suggests that NETosis might not be dependent on a specific lipopeptide sequence. Also, NETosis is partially abolished when TLR2 is blocked with specific antibodies. The results presented in this work provide evidences for the mechanisms underlying NET activation in mycoplasma infections, and on their contribution to pathogenesis of mycoplasmosis.
Asunto(s)
Proteínas Bacterianas/farmacología , Trampas Extracelulares/química , Lipoproteínas/farmacología , Glándulas Mamarias Animales/inmunología , Mycoplasma agalactiae/química , Neutrófilos/efectos de los fármacos , Animales , Anticuerpos Neutralizantes/farmacología , Proteínas Bacterianas/síntesis química , Membrana Celular/química , Membrana Celular/inmunología , Trampas Extracelulares/inmunología , Trampas Extracelulares/metabolismo , Femenino , Expresión Génica , Lipopolisacáridos/farmacología , Lipoproteínas/síntesis química , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/microbiología , Leche/inmunología , Leche/microbiología , Mycoplasma agalactiae/inmunología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/microbiología , Cultivo Primario de Células , Ovinos , Acetato de Tetradecanoilforbol/farmacología , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunologíaRESUMEN
Synthetic lipoproteins represent a relevant tool for targeted delivery of biological/chemical agents (chemotherapeutics, siRNAs, photosensitizers, and imaging contrast agents) into various cell types. These nanoparticles offer a number of advantages for drugs delivery over their native counterparts while retaining their natural characteristics and biological functions. Their ultra-small size (<30 nm), high biocompatibility, favorable circulation half-life, and natural ability to bind specific lipoprotein receptors, i.e., low-density lipoprotein receptor (LDLR) and Scavenger receptor class B member 1 (SRB1) that are found in a number of pathological conditions (e.g., cancer, atherosclerosis), make them superior delivery strategies when compared with other nanoparticle systems. We review the various approaches that have been developed for the generation of synthetic lipoproteins and their respective applications in vitro and in vivo. More specifically, we summarize the approaches employed to address the limitation on use of reconstituted lipoproteins by means of natural or recombinant apolipoproteins, as well as apolipoprotein mimetic molecules. Finally, we provide an overview of the advantages and disadvantages of these approaches and discuss future perspectives for clinical translation of these nanoparticles.
Asunto(s)
Materiales Biomiméticos/síntesis química , Medios de Contraste/síntesis química , Lipoproteínas/síntesis química , Lipoproteínas/farmacocinética , Terapia Molecular Dirigida/métodos , Nanocápsulas/química , Animales , Cristalización/métodos , Humanos , Nanocápsulas/administración & dosificaciónRESUMEN
There is currently no Lyme borreliosis vaccine available for humans, although it has been shown that the disease can be prevented by immunization with an OspA-based vaccine (LYMErix). Outer surface protein A (OspA) is one of the dominant antigens expressed by the spirochetes when present in a tick. The Borrelia species causing Lyme borreliosis in Europe express different OspA serotypes on their surface, B. burgdorferi (serotype 1), B. afzelii (serotype 2), B. garinii (serotypes, 3, 5 and 6) and B. bavariensis (serotype 4), while only B. burgdorferi is present in the US. In order to target all these pathogenic Borrelia species, we have designed a multivalent OspA-based vaccine. The vaccine includes three proteins, each containing the C-terminal half of two OspA serotypes linked to form a heterodimer. In order to stabilize the C-terminal fragment and thus preserve important structural epitopes at physiological temperature, disulfide bonds were introduced. The immunogenicity was increased by introduction of a lipidation signal which ensures the addition of an N-terminal lipid moiety. Three immunizations with 3.0 µg adjuvanted vaccine protected mice from a challenge with spirochetes expressing either OspA serotype 1, 2 or 5. Mice were protected against both challenge with infected ticks and in vitro grown spirochetes. Immunological analyses (ELISA, surface binding and growth inhibition) indicated that the vaccine can provide protection against the majority of Borrelia species pathogenic for humans. This article presents the approach which allows for the generation of a hexavalent vaccine that can potentially protect against a broad range of globally distributed Borrelia species causing Lyme borreliosis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/síntesis química , Vacunas Bacterianas/síntesis química , Borrelia/inmunología , Lipoproteínas/síntesis química , Vacunas contra Enfermedad de Lyme/síntesis química , Enfermedad de Lyme/prevención & control , Animales , Antígenos de Superficie/química , Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/química , Vacunas Bacterianas/inmunología , Borrelia/efectos de los fármacos , Modelos Animales de Enfermedad , Epítopos/inmunología , Femenino , Humanos , Lipoproteínas/química , Lipoproteínas/inmunología , Enfermedad de Lyme/inmunología , Vacunas contra Enfermedad de Lyme/administración & dosificación , Ratones , Ratones Endogámicos C3H , Garrapatas/microbiologíaRESUMEN
Multivalent synthetic vaccines were obtained by solid-phase synthesis of tumor-associated MUC1 glycopeptide antigens and their coupling to a Pam3 Cys lipopeptide through click reactions. These vaccines elicited immune responses in mice without the use of any external adjuvant. The vaccine containing four copies of a MUC1 sialyl-TN antigen showed a significant cluster effect. It induced in mice prevailing IgG2a antibodies, which bind to MCF-7 breast tumor cells and initiate the killing of these tumor cells by activation of the complement-dependent cytotoxicity complex.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Glicopéptidos/química , Lipoproteínas/química , Mucina-1/química , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Vacunas contra el Cáncer/síntesis química , Vacunas contra el Cáncer/química , Química Clic , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Glicopéptidos/síntesis química , Humanos , Lipoproteínas/síntesis química , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Mucina-1/inmunología , Mucina-1/metabolismo , ConejosRESUMEN
Using TLR agonists in cancer treatment can have either beneficial or detrimental effects. Therefore, it is important to determine their effect on the tumor growth and understand the underlying mechanisms in animal tumor models. In this study, we report a general immunotherapeutic activity of a synthetic bacterial lipoprotein (BLP), a TLR1/TLR2 agonist, on established lung carcinoma, leukemia, and melanoma in mice. Systemic treatment of 3LL tumor-bearing mice with BLP, but not LPS, led to a dose-dependent tumor regression and a long-lasting protective response against tumor rechallenge. The BLP-mediated tumor remission was neither mediated by a direct tumoricidal activity nor by innate immune cells, because it lacked therapeutic effect in immunodeficient SCID mice. Instead, BLP treatment reduced the suppressive function of Foxp3(+) regulatory T cells (Tregs) and enhanced the cytotoxicity of tumor-specific CTL in vitro and in vivo. Furthermore, adoptive cotransfer of BLP-pretreated but not untreated CTL and Tregs from wild-type but not from TLR2(-/-) mice was sufficient to restore antitumor immunity in SCID mice by reciprocally modulating Treg and CTL function. These results demonstrate that the TLR1/TLR2 agonist BLP may have a general tumor therapeutic property involving reciprocal downregulation of Treg and upregulation of CTL function. This property may play an important role in the development of novel antitumor strategies.
Asunto(s)
Carcinoma Pulmonar de Lewis/prevención & control , Leucemia Experimental/prevención & control , Melanoma Experimental/prevención & control , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Receptor Toll-Like 1/agonistas , Receptor Toll-Like 2/agonistas , Animales , Antineoplásicos/agonistas , Antineoplásicos/síntesis química , Proteínas Bacterianas/síntesis química , Proteínas Bacterianas/uso terapéutico , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/patología , Regulación hacia Abajo/inmunología , Humanos , Leucemia Experimental/inmunología , Leucemia Experimental/patología , Lipoproteínas/síntesis química , Lipoproteínas/uso terapéutico , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Noqueados , Ratones SCID , Linfocitos T Citotóxicos/patología , Linfocitos T Citotóxicos/trasplante , Linfocitos T Reguladores/patología , Linfocitos T Reguladores/trasplante , Receptor Toll-Like 1/fisiología , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/fisiología , Regulación hacia Arriba/inmunologíaRESUMEN
There is currently a need for improved serological tests for the diagnosis and monitoring of Lyme disease, an infection caused by Borrelia burgdorferi. In the present study, we evaluated luciferase immunoprecipitation systems (LIPSs) for use for profiling of the antibody responses to a panel of B. burgdorferi proteins for the diagnosis of Lyme disease. Initially, serum samples from a cohort of patients and controls (n = 46) were used for training and were profiled by the use of 15 different B. burgdorferi antigen constructs. For the patient sera, the antibody responses to several B. burgdorferi antigens, including VlsE, flagellin (FlaB), BmpA, DbpA, and DbpB, indicated that the antigens had high levels of immunoreactivity. However, the best diagnostic performance was achieved with a synthetic protein, designated VOVO, consisting of a repeated antigenic peptide sequence, VlsE-OspC-VlsE-OspC, Analysis of an independent set of serum samples (n = 139) used for validation showed that the VOVO LIPS test had 98% sensitivity (95% confidence interval [CI], 93% to 100%; P < 0.0001) and 100% specificity (95% CI, 94% to 100%; P < 0.0001). Similarly, the C6 peptide enzyme-linked immunosorbent assay (ELISA) also had 98% sensitivity (95% CI, 93% to 100%; P < 0.0001) and 98% specificity (95% CI, 90% to 100%; P < 0.0001). Receiver operating characteristic analysis revealed that the rates of detection of Lyme disease by the LIPS test and the C6 ELISA were not statistically different. However, the VOVO LIPS test displayed a wide dynamic range of antibody detection spanning over 10,000-fold without the need for serum dilution. These results suggest that screening by the LIPS test with VOVO and other B. burgdorferi antigens offers an efficient quantitative approach for evaluation of the antibody responses in patients with Lyme disease.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos , Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas , Borrelia burgdorferi/inmunología , Lipoproteínas , Enfermedad de Lyme/diagnóstico , Péptidos/inmunología , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/síntesis química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/síntesis química , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Humanos , Inmunoprecipitación , Lipoproteínas/síntesis química , Lipoproteínas/química , Lipoproteínas/inmunología , Luciferasas/genética , Luciferasas/metabolismo , Enfermedad de Lyme/inmunología , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Curva ROC , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Factores de TiempoRESUMEN
Biologically functional Ras isoforms undergo post-translational modifications starting with farnesylation of the most C-terminal cysteine. Combined with further processing steps, this isoprenylation allows for the anchoring of these proteins in endomembranes, where signal transduction events take place. The specific localization is subject to dynamic regulation and assumed to modulate the activity of Ras proteins by governing their spatiotemporal distribution. The delta subunit of phosphodiesterase (PDEdelta) has attracted attention as a solubilization factor of isoprenylated Ras. In this study, we demonstrate that critical residues in the putative isoprenoid pocket of PDEdelta can be mapped by coupling with a semisynthetic N-Ras lipoprotein in which the native farnesyl group of the processed protein was replaced by a photoactivatable geranyl benzophenone moiety. The crosslinked product included parts of beta-sheet 9 of PDEdelta, which contains the highly conserved amino acids V145 and L147. Modeling of the PDEdelta-geranyl benzophenone (GerBP) complex supports the conclusion that the photolabeled sequence is embedded in the putative isoprenoid pocket of PDEdelta.
Asunto(s)
Luz , Lipoproteínas/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Terpenos/metabolismo , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Biología Computacional , Reactivos de Enlaces Cruzados/metabolismo , Humanos , Lipoproteínas/síntesis química , Lipoproteínas/química , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas ras/síntesis química , Proteínas ras/químicaRESUMEN
Matrix metalloproteinases (MMPs) constitute a class of extracellular-matrix-degrading enzymes overexpressed in many cancers and contribute to the metastatic ability of the cancer cells. We have recently demonstrated that liposomal contents can be released when triggered by the enzyme MMP-9. Herein, we report the results of our mechanistic studies of the MMP-9-triggered release of liposomal contents. We synthesized peptides containing the cleavage site for MMP-9 and conjugated them with fatty acids to prepare the corresponding lipopeptides. By employing circular dichroism (CD) spectroscopy, we demonstrated that the lipopeptides, when incorporated into liposomes, are demixed in the lipid bilayers and generate triple-helical structures. MMP-9 cleaves the triple-helical peptides, leading to the release of the liposomal contents. Other MMPs, which cannot hydrolyze triple-helical peptides, fail to release the contents from the liposomes. We also observed that the rate and extent of release of the liposomal contents depend on the mismatch between the acyl chains of the synthesized lipopeptide and phospholipid components of the liposomes. CD spectroscopic studies imply that the observed differences in the release reflect the ability of the liposomal membrane to anneal the defects following the enzymatic cleavage of the liposome-incorporated lipopeptides.
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Lipoproteínas/química , Liposomas/química , Metaloproteinasa 9 de la Matriz/química , Dicroismo Circular , Fluoresceínas/química , Humanos , Hidrólisis , Lipoproteínas/síntesis química , Péptidos/síntesis química , Péptidos/química , Estructura Secundaria de ProteínaRESUMEN
SUMMARY: Serum-free culture conditions to generate immature human monocyte-derived DC (Mo-DC) were optimized, and the parameters that influence their maturation after exposure to lipopeptides containing CD4(+) and CD8(+) T-cell epitopes were examined. The lipopeptides contained a single CD4(+) helper T-cell epitopes, one of a number of human leucocyte antigen (HLA)-A2-restricted cytotoxic T-cell epitope and the lipid Pam2Cys. To ensure complete maturation of the Mo-DC, we examined (i) the optimal lipopeptide concentration, (ii) the optimal Mo-DC density and (iii) the appropriate period of exposure of the Mo-DC to the lipopeptides. The results showed that a high dose of lipopeptide (30 microm) was no more efficient at upregulating maturation markers on Mo-DC than a low dose (6 microm). There was an inverse relationship between Mo-DC concentration and the mean fluorescence intensity of maturation markers. In addition, at the higher cell concentrations, the chemotactic capacity of the Mo-DC towards a cognate ligand, CCL21, was reduced. Thus, high cell concentrations during lipopeptide exposure were detrimental to Mo-DC maturation and function. The duration of exposure of Mo-DC to the lipopeptides had little effect on phenotype, although Mo-DC exposed to lipopeptides for 48 rather than 4 h showed an increased ability to stimulate autologous peripheral blood mononuclear cells to release interferon-gamma in the absence of exogenous maturation factors. These findings reveal conditions for generating mature antigen-loaded DC suitable for targeted immunotherapy.
Asunto(s)
Células Dendríticas/citología , Células Dendríticas/inmunología , Lipoproteínas/inmunología , Activación de Linfocitos , Péptidos/inmunología , Linfocitos T/inmunología , Adulto , Secuencia de Aminoácidos , Diferenciación Celular , Técnicas de Cocultivo , Medio de Cultivo Libre de Suero , Células Dendríticas/efectos de los fármacos , Epítopos de Linfocito T/química , Humanos , Memoria Inmunológica , Lipoproteínas/síntesis química , Lipoproteínas/química , Masculino , Persona de Mediana Edad , Monocitos/citología , Péptidos/síntesis química , Péptidos/química , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The inefficiency of antifungal agents and high mortality rate resulting from invasive aspergillosis remain major clinical concerns. Recently, we reported on a new family of ultrashort cationic lipopeptides active in vitro against fungi. Mode of action studies supported a membranolytic or a detergent-like effect. Here, we screened several lipopeptides in vitro for their anti-A. fumigatus activity. To investigate the therapeutic properties of the selected peptides in vivo, we challenged immunosuppressed C57BL/6 wild-type mice intranasally with DsRed-labeled A. fumigatus conidia and subsequently treated the animals locally with the lipopeptides. Confocal microscopic analysis revealed the degradation of DsRed-labeled hyphal forms and residual conidia in the lungs of the mice. The most efficient peptide was tested further using a survival assay and was found to significantly prolong the life of the treated animals, whereas no mice survived with the current standard antifungal treatment with amphotericin B. Moreover, as opposed to the drug-treated lungs, the peptide-treated lungs did not display any toxicity of the peptide. Our results highlight the potential of this family of lipopeptides for the treatment of pulmonary invasive aspergillosis.