RESUMEN

BACKGROUND: Skin, the largest organ of the human body by weight, hosts a diversity of microorganisms that can influence health. The microbial residents of the skin are now appreciated for their roles in host immune interactions, wound healing, colonization resistance, and various skin disorders. Still, much remains to be discovered in terms of the host pathways influenced by skin microorganisms, as well as the higher-level skin properties impacted through these microbe-host interactions. Towards this direction, recent efforts using mouse models point to pronounced changes in the transcriptional profiles of the skin in response to the presence of a microbial community. However, there is a need to quantify the roles of microorganisms at both the individual and community-level in healthy human skin. In this study, we utilize human skin equivalents to study the effects of individual taxa and a microbial community in a precisely controlled context. Through transcriptomics analysis, we identify key genes and pathways influenced by skin microbes, and we also characterize higher-level impacts on skin processes and properties through histological analyses. RESULTS: The presence of a microbiome on a 3D skin tissue model led to significantly altered patterns of gene expression, influencing genes involved in the regulation of apoptosis, proliferation, and the extracellular matrix (among others). Moreover, microbiome treatment influenced the thickness of the epidermal layer, reduced the number of actively proliferating cells, and increased filaggrin expression. Many of these findings were evident upon treatment with the mixed community, but either not detected or less pronounced in treatments by single microorganisms, underscoring the impact that a diverse skin microbiome has on the host. CONCLUSIONS: This work contributes to the understanding of how microbiome constituents individually and collectively influence human skin processes and properties. The results show that, while it is important to understand the effect of individual microbes on the host, a full community of microbes has unique and pronounced effects on the skin. Thus, in its impacts on the host, the skin microbiome is more than the sum of its parts. Video abstract.

Asunto(s)

Interacciones Microbiota-Huesped , Microbiota , Fenómenos Fisiológicos de la Piel , Piel/metabolismo , Piel/microbiología , Proteínas Filagrina , Perfilación de la Expresión Génica , Voluntarios Sanos , Interacciones Microbiota-Huesped/genética , Humanos , Microbiota/genética , Fenómenos Fisiológicos de la Piel/genética

RESUMEN

A review of nasal sprays and gargles with antiviral properties suggests that a number of commonly used antiseptics including povidone-iodine, Listerine®, iota-carrageenan and chlorhexidine should be studied in clinical trials to mitigate both the progression and transmission of SARS-CoV-2. Several of these antiseptics have demonstrated the ability to cut the viral load of SARS-CoV-2 by 3-4 log10 in 15-30 s in vitro. In addition, hypertonic saline targets viral replication by increasing hypochlorous acid inside the cell. A number of clinical trials are in process to study these interventions both for prevention of transmission, prophylaxis after exposure, and to diminish progression by reduction of viral load in the early stages of infection.

Asunto(s)

Antiinfecciosos Locales/administración & dosificación , Antiinfecciosos Locales/uso terapéutico , COVID-19/prevención & control , SARS-CoV-2/efectos de los fármacos , COVID-19/transmisión , Carragenina/uso terapéutico , Clorhexidina/uso terapéutico , Combinación de Medicamentos , Peróxido de Hidrógeno/uso terapéutico , Rociadores Nasales , Aceites Volátiles/uso terapéutico , Povidona Yodada/uso terapéutico , Salicilatos/uso terapéutico , Terpenos/uso terapéutico , Carga Viral/efectos de los fármacos

RESUMEN

BACKGROUND: The skin micro-environment varies across the body, but all sites are host to microorganisms that can impact skin health. Some of these organisms are true commensals which colonize a unique niche on the skin, while open exposure of the skin to the environment also results in the transient presence of diverse microbes with unknown influences on skin health. Culture-based studies of skin microbiota suggest that skin microbes can affect skin properties, immune responses, pathogen growth, and wound healing. RESULTS: In this work, we greatly expanded the diversity of available commensal organisms by collecting > 800 organisms from 3 body sites of 17 individuals. Our collection includes > 30 bacterial genera and 14 fungal genera, with Staphylococcus and Micrococcus as the most prevalent isolates. We characterized a subset of skin isolates for the utilization of carbon compounds found on the skin surface. We observed that members of the skin microbiota have the capacity to metabolize amino acids, steroids, lipids, and sugars, as well as compounds originating from personal care products. CONCLUSIONS: This collection is a resource that will support skin microbiome research with the potential for discovery of novel small molecules, development of novel therapeutics, and insight into the metabolic activities of the skin microbiota. We believe this unique resource will inform skin microbiome management to benefit skin health. Video abstract.

Asunto(s)

Bacterias , Hongos , Microbiota , Piel/microbiología , Adolescente , Adulto , Bacterias/clasificación , Bacterias/aislamiento & purificación , Hongos/clasificación , Hongos/aislamiento & purificación , Voluntarios Sanos , Humanos , Persona de Mediana Edad , Adulto Joven

RESUMEN

The characterization of innate immune activation is crucial for vaccine and therapeutic development, including RNA-based vaccines, a promising approach. Current measurement methods quantify type I interferon and inflammatory cytokine production, but they do not allow for the isolation of individual pathways, do not provide kinetic activation or spatial information within tissues, and cannot be translated into clinical studies. Here we demonstrated the use of proximity ligation assays (PLAs) to detect pattern recognition receptor (PRR) activation in cells and in tissue samples. First, we validated PLA's sensitivity and specificity using well-characterized soluble agonists. Next, we characterized PRR activation from in vitro-transcribed (IVT) mRNAs, as well as the effect of sequence and base modifications in vitro. Finally, we established the measurement of PRR activation in tissue sections via PLA upon IVT mRNA intramuscular (i.m.) injection in mice. Overall, our results indicate that PLA is a valuable, versatile, and sensitive tool to monitor PRR activation for vaccine, adjuvant, and therapeutic screening.

RESUMEN

In vitro transcribed (IVT) mRNA is an appealing platform for next generation vaccines, as it can be manufactured rapidly at large scale to meet emerging pathogens. However, its performance as a robust vaccine is strengthened by supplemental immune stimulation, which is typically provided by adjuvant formulations that facilitate delivery and stimulate immune responses. Here, we present a strategy for increasing translation of a model IVT mRNA vaccine while simultaneously modulating its immune-stimulatory properties in a programmable fashion, without relying on delivery vehicle formulations. Substitution of uridine with the modified base N1-methylpseudouridine reduces the intrinsic immune stimulation of the IVT mRNA and enhances antigen translation. Tethering adjuvants to naked IVT mRNA through antisense nucleotides boosts the immunostimulatory properties of adjuvants in vitro, without impairing transgene production or adjuvant activity. In vivo, intramuscular injection of tethered IVT mRNA-TLR7 agonists leads to enhanced local immune responses, and to antigen-specific cell-mediated and humoral responses. We believe this system represents a potential platform compatible with any adjuvant of interest to enable specific programmable stimulation of immune responses.

Asunto(s)

Inmunidad Innata/efectos de los fármacos , ARN Mensajero/genética , Vacunas Sintéticas/farmacología , Animales , Formación de Anticuerpos , Inmunidad Celular , Inyecciones Intramusculares , Ratones , Células RAW 264.7 , Transcripción Genética , Vacunas Sintéticas/administración & dosificación

RESUMEN

The translational efficiency of an in vitro transcribed (IVT) mRNA was measured upon delivery to primary skeletal muscle cells and to a mouse model system, towards the development of a predictive in vitro assay for the screening and validation of intramuscular mRNA-based vaccines. When IVT mRNA was delivered either naked or complexed with novel aminoglycoside-based delivery vehicles, significant differences in protein expression in vitro and in vivo were observed. We hypothesized that this previously anticipated discrepancy was due to differences in the mechanism of IVT mRNA endosomal entry and release following delivery. To address this, IVT mRNA was fluorescently labeled prior to delivery, to visualize its distribution. Colocalization with endosomal markers indicated that different entry pathways were utilized in vivo and in vitro, depending on the delivery vehicle, resulting in variations in protein expression levels. Since extracellular matrix stiffness (ECM) influences mRNA entry, trafficking and release, the effect of mechanotransduction on mRNA expression was investigated in vitro upon delivery of IVT mRNA alone, and complexed with delivery vehicles to skeletal muscle cells grown on ∼10 kPa hydrogels. This in vitro hydrogel model more accurately recapitulated the results obtained in vivo upon IM injection, indicating that this approach may assist in the characterization of mRNA based vaccines.

Asunto(s)

Mecanotransducción Celular/fisiología , Músculo Esquelético/metabolismo , ARN Mensajero/metabolismo , Animales , Línea Celular , Endosomas/química , Matriz Extracelular/química , Femenino , Citometría de Flujo , Células HeLa , Humanos , Hidrogeles/química , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química

RESUMEN

The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections, which will facilitate mRNA trafficking studies in pre-clinical models. Overall, we demonstrate the ability to study fundamental mechanisms necessary to optimize delivery and therapeutic strategies, in order to design the next generation of novel mRNA therapeutics and vaccines.

Asunto(s)

Endosomas/metabolismo , Imagen Óptica/métodos , ARN Mensajero/farmacocinética , Análisis de la Célula Individual/métodos , Coloración y Etiquetado/métodos , Animales , Transporte Biológico , Carbocianinas/química , Línea Celular , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Fibroblastos/citología , Fibroblastos/metabolismo , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Rayos Infrarrojos , Inyecciones Intramusculares , Ratones , Sondas Moleculares/química , Hibridación de Ácido Nucleico , ARN Mensajero/química , ARN Mensajero/genética

RESUMEN

Synthetic mRNA has recently shown great potential as a tool for genetic introduction of proteins. Its utility as a gene carrier has been demonstrated in several studies for both the introduction of therapeutic proteins and subunit vaccines. At one point, synthetic mRNA was believed to be too immunogenic and labile for pharmaceutical purposes. However, the development of several strategies have enabled mRNA technology to overcome these challenges, including incorporation of modified nucleotides, codon optimization of the coding region, incorporation of untranslated regions into the mRNA, and the use of delivery vehicles. While these approaches have been shown to enhance performance of some mRNA constructs, gene-to-gene variation and low efficiency of mRNA protein production are still significant hurdles. Further mechanistic understanding of how these strategies affect protein production and innate immune activation is needed for the widespread adoption for both therapeutic and vaccine applications. This review highlights key studies involved in the development of strategies employed to increase protein expression and control the immunogenicity of synthetic mRNA. Areas in the literature where improved understanding is needed will also be discussed.

RESUMEN

Spinal cord injury (SCI) can lead to permanent motor and sensory deficits. Following the initial traumatic insult, secondary injury mechanisms characterized by persistent heightened inflammation are initiated and lead to continued and pervasive cell death and tissue damage. Anti-inflammatory drugs such as methylprednisolone (MP) used clinically have ambiguous benefits with debilitating side effects. Typically, these drugs are administered systemically at high doses, resulting in toxicity and paradoxically increased inflammation. Furthermore, these drugs have a small time window postinjury (few hours) during which they need to be infused to be effective. As an alternative to MP, we investigated the effect of a small molecule inhibitor (Chicago sky blue, CSB) of macrophage migration inhibitory factor (MIF) for treating SCI. The pleiotropic cytokine MIF is known to contribute to upregulation of several pro-inflammatory cytokines in various disease and injury states. In vitro, CSB administration alleviated endotoxin-mediated inflammation in primary microglia and macrophages. Nanocarriers such as liposomes can potentially alleviate systemic side effects of high-dose therapy by enabling site-specific drug delivery to the spinal cord. However, the therapeutic window of 100 nm scale nanoparticle localization to the spinal cord after contusion injury is not fully known. Thus, we first investigated the ability of nanocarriers of different sizes to localize to the injured spinal cord up to 2 weeks postinjury. Results from the study showed that nanocarriers as large as 200 nm in diameter could extravasate into the injured spinal cord up to 96 h postinjury. We then formulated nanocarriers (liposomes) encapsulating CSB and administered them intravenously 48 h postinjury, within the previously determined 96 h therapeutic window. In vivo, in this clinically relevant contusion injury model in rats, CSB administration led to preservation of vascular and white matter integrity, improved wound healing, and an increase in levels of arginase and other transcripts indicative of a resolution phase of wound healing. This study demonstrates the potential of MIF inhibition in SCI and the utility of nanocarrier-mediated drug delivery selectively to the injured cord.

Asunto(s)

Portadores de Fármacos/química , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Nanoestructuras/química , Traumatismos de la Médula Espinal/complicaciones , Azul de Tripano/química , Azul de Tripano/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Liposomas , Masculino , Ratones , Tamaño de la Partícula , Polietilenglicoles/química , Células RAW 264.7 , Ratas , Factores de Tiempo

RESUMEN

Human herpesvirus 6B (HHV-6B) is the causative agent of roseola infantum. HHV-6A and 6B can reactivate in immunosuppressed individuals and are linked with severe inflammatory response, organ rejection and central nervous system diseases. About 0.85% of the US and UK population carries an integrated HHV-6 genome in all nucleated cells through germline transmission. We have previously reported that the HHV-6A genome integrated in telomeres of patients suffering from neurological dysfunction and also in telomeres of tissue culture cells. We now report that HHV-6B also integrates in telomeres during latency. Detailed mapping of the integrated viral genomes demonstrates that a single HHV-6 genome integrates and telomere repeats join the left end of the integrated viral genome. When HEK-293 cells carrying integrated HHV-6A were exposed to the histone deacetylase inhibitor Trichostatin A, circularization and/or formation of concatamers were detected and this assay could be used to distinguish between lytic replication and latency.

Asunto(s)

Mapeo Cromosómico , Genoma Viral , Herpesvirus Humano 6/genética , Telómero/virología , Integración Viral , Línea Celular , Cromosomas Humanos/virología , Replicación del ADN , ADN Viral/genética , Femenino , Células HEK293/efectos de los fármacos , Células HEK293/virología , Humanos , Ácidos Hidroxámicos/farmacología , Masculino , Infecciones por Roseolovirus/virología , Latencia del Virus

RESUMEN

BACKGROUND: Human herpesvirus 6 (HHV-6) is a neurotropic virus implicated in central nervous system (CNS) dysfunction, multiple sclerosis, seizures and encephalitis. Inherited or "chromosomally integrated" HHV-6 (CIHHV-6) is a condition characterized by high DNA loads and germ line transmission of HHV-6 genomes, which are integrated into the telomere. OBJECTIVES: We previously reported that integrated HHV-6 can be reactivated by trichostatin A in vitro. Therefore, we hypothesized that a broad array of neurological symptoms of CIHHV-6 patients may respond to antiviral drug treatment. STUDY DESIGN: The patients have been treated with antiviral drugs and monitored for viral load, late mRNA, and clinical improvement. RESULTS: Antiviral therapy of two CIHHV patients resulted in successful clinical resolution. However, both patients relapsed on multiple occasions within 4-6 months of cessation of antiviral therapy. CONCLUSIONS: Successful antiviral drug treatment suggests that clinical symptoms of these patients were due to symptomatic reactivation of CIHHV-6. Alternatively, some CIHHV-6 patients may have a reduced resistance to community-acquired HHV-6 strains due to tolerance leading to persistent infections.

Asunto(s)

Antivirales/uso terapéutico , Trastornos del Conocimiento/virología , Herpesvirus Humano 6/genética , Infecciones por Roseolovirus/tratamiento farmacológico , Integración Viral , Niño , ADN Viral/sangre , Electroencefalografía , Femenino , Humanos , Recuento de Leucocitos , Masculino , ARN Mensajero/sangre , Infecciones por Roseolovirus/genética , Infecciones por Roseolovirus/psicología , Infecciones por Roseolovirus/virología , Hermanos , Carga Viral , Adulto Joven

RESUMEN

Chromosomally integrated human herpesvirus 6 (ciHHV-6) is a condition in which the complete HHV-6 genome is integrated into the host germ line genome and is vertically transmitted in a Mendelian manner. The condition is found in less than 1% of controls in the USA and UK, but has been found at a somewhat higher prevalence in transplant recipients and other patient populations in several small studies. HHV-6 levels in whole blood that exceed 5.5 log10 copies/ml are strongly suggestive of ciHHV-6. Monitoring DNA load in plasma and serum is unreliable, both for identifying and for monitoring subjects with ciHHV-6 due to cell lysis and release of cellular DNA. High HHV-6 DNA loads associated with ciHHV-6 can lead to erroneous diagnosis of active infection. Transplant recipients with ciHHV-6 may be at increased risk for bacterial infection and graft rejection. ciHHV-6 can be induced to a state of active viral replication in vitro. It is not known whether ciHHV-6 individuals are put at clinical risk by the use of drugs that have been associated with HHV-6 reactivation in vivo or in vitro. Nonetheless, we urge careful observation when use of such drugs is indicated in individuals known to have ciHHV-6. Little is known about whether individuals with ciHHV-6 develop immune tolerance for viral proteins. Further research is needed to determine the role of ciHHV-6 in disease.

Asunto(s)

Cromosomas Humanos/virología , Herpesvirus Humano 6/fisiología , Infecciones por Roseolovirus/virología , Integración Viral , Herpesvirus Humano 6/genética , Humanos , Infecciones por Roseolovirus/genética

RESUMEN

We previously reported the formulation and physical properties of HER2 (human epidermal growth factor receptor 2)-specific affibody (ZHER2:342-Cys) conjugated thermosensitive liposomes (HER2(+)affisomes). Here we examined localized delivery potential of these affisomes by monitoring cellular interactions, intracellular uptake, and hyperthermia-induced effects on drug delivery. We modified ZHER2:342-Cys by introducing a glycine-serine spacer before the C-terminus cysteine (called ZHER2-GS-Cys) to achieve accessibility to cell surface expressed HER2. This modification did not affect HER2-specific binding and ZHER2-GS-Cys retained its ability to conjugate to the liposomes containing dipalmitoyl phosphatidyl choline: DSPE-PEG2000-Malemide, 96:04 mole ratios (HER2(+)affisomes). HER2(+)affisomes were either (i) fluorescently labeled with rhodamine-PE and calcein or (ii) loaded with an anticancer drug doxorubicin (DOX). Fluorescently labeled HER2(+) affisomes showed at least 10-fold increase in binding to HER2(+) cells (SK-BR-3) when compared to HER2(-) cells (MDA-MB-468) at 37°C. A competition experiment using free ZHER2-GS-Cys blocked HER2(+) affisome-SK-BR-3 cell associations. Imaging with confocal microscopy showed that HER2(+) affisomes accumulated in the cytosol of SK-BR-3 cells at 37°C. Hyperthermia-induced intracellular release experiments showed that the treatment of HER2(+) affisome/SK-BR-3 cell complexes with a 45°C (±1°C) pre-equilibrated buffer resulted in cytosolic delivery of calcein. Substantial calcein release was observed within 20min at 45°C, with no effect on cell viability under these conditions. Similarly, DOX-loaded HER2(+)affisomes showed at least 2- to 3-fold higher accumulation of DOX in SK-BR-3 cells as compared to control liposomes. DOX-mediated cytotoxicity was more pronounced in SK-BR-3 cells especially at lower doses of HER2(+)affisomes. Brief exposure of liposome-cell complexes at 45°C prior to the onset of incubations for cell killing assays resulted in enhanced cytotoxicity for affisomes and control liposomes. However, Doxil (a commercially available liposome formulation) showed significantly lower toxicity under identical conditions. Therefore, our data demonstrate that HER2(+)affisomes encompass both targeting and triggering potential and hence may prove to be viable nanodrug delivery carriers for breast cancer treatment.

Asunto(s)

Antibióticos Antineoplásicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos , Liposomas/metabolismo , Receptor ErbB-2/metabolismo , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Femenino , Humanos , Hipertermia Inducida , Liposomas/química

RESUMEN

The CD22 antigen is a viable target for therapeutic intervention for B-cell lymphomas. Several therapeutic anti-CD22 antibodies as well as an anti-CD22-based immunotoxin (HA22) are currently under investigation in clinical settings. Coupling of anti-CD22 reagents with a nano-drug delivery vehicle is projected to significantly improve treatment efficacies. Therefore, we generated a mutant of the targeting segment of HA22 (a CD22 scFv) to increase its soluble expression (mut-HA22), and conjugated it to the surface of sonicated liposomes to generate immunoliposomes (mut-HA22-liposomes). We examined liposome binding and uptake by CD22(+) B-lymphocytes (BJAB) by using calcein and/or rhodamine PE-labeled liposomes. We also tested the effect of targeting on cellular toxicity with doxorubicin-loaded liposomes. We report that: (i) Binding of mut-HA22-liposomes to BJAB cells was significantly greater than liposomes not conjugated with mut-HA22 (control liposomes), and mut-HA22-liposomes bind to and are taken in by BJAB cells in a dose and temperature-dependent manner, respectively; (ii) This binding occurred via the interaction with the cellular CD22 as pre-incubation of the cells with mut-HA22 blocked subsequent liposome binding; (iii) Intracellular localization of mut-HA22-liposomes at 37 degrees C but not at 4 degrees C indicated that our targeted liposomes were taken up through an energy dependent process via receptor-mediated endocytosis; and (iv) Mut-HA22-liposomes loaded with doxorubicin exhibited at least 2-3 fold more accumulation of doxorubicin in BJAB cells as compared to control liposomes. Moreover, these liposomes showed at least a 2-4 fold enhanced killing of BJAB or Raji cells (CD22(+)), but not SUP-T1 cells (CD22(-)). Taken together these data suggest that these 2nd-generation liposomes may serve as promising carriers for targeted drug delivery to treat patients suffering from B-cell lymphoma.

Asunto(s)

Linfocitos B/metabolismo , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Anticuerpos de Cadena Única/inmunología , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Línea Celular , Supervivencia Celular , Doxorrubicina/farmacología , Citometría de Flujo , Colorantes Fluorescentes , Humanos , Liposomas/sangre , Liposomas/metabolismo , Nanopartículas , Fosfatidiletanolaminas/metabolismo , Lectina 2 Similar a Ig de Unión al Ácido Siálico/efectos de los fármacos , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo

RESUMEN

For multiple site-specific conjugations of bioactive molecules to a single-chain antibody (scFv) molecule, we have constructed a human anti HER2 receptor, scFv, with a C-terminal fusion polypeptide containing 1, 3, or 17 threonine (Thr) residues. The C-terminal extended fusion polypeptides of these recombinant scFv fusion proteins are used as the acceptor substrate for human polypeptide-alpha-Nu-acetylgalactosaminyltransferase II (h-ppGalNAc-T2) that transfers either GalNAc or 2-keto-Gal, a modified galactose with a chemical handle, from their respective UDP-sugars to the side-chain hydroxyl group of the Thr residue(s). The recombinant scFv fusion proteins are expressed in E. coli as inclusion bodies and in vitro refolded and glycosylated with h-ppGalNAc-T2. Upon protease cleavage, the MALDI-TOF spectra of the glycosylated C-terminal fusion polypeptides showed that the glycosylated scFv fusion protein with a single Thr residue is fully glycosylated with a single 2-keto-Gal, whereas the glycosylated scFv fusion protein with 3 and 17 Thr residues is found as an equal mixture of 2-3 and 5-8 2-keto-Gal glycosylated fusion proteins, respectively. These fusion scFv proteins with the modified galactose are then conjugated with a fluorescence probe, Alexa488, that carries an orthogonal reactive group. The fluorescence labeled scFv proteins bind specifically to a human breast cancer cell line (SK-BR-3) that overexpresses the HER2 receptor, indicating that the in vitro folded scFv fusion proteins are biologically active and the presence of conjugated multiple Alexa488 probes in their C-terminal end does not interfere with their binding to the antigen.

Asunto(s)

Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Colorantes Fluorescentes/química , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Escherichia coli/genética , Expresión Génica , Glicosilación , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Receptor ErbB-2/genética

RESUMEN

The Fc N-glycan chains of four therapeutic monoclonal antibodies (mAbs), namely, Avastin, Rituxan, Remicade, and Herceptin, released by PNGase F, show by MALDI analysis that these biantennary N-glycans are a mixture of G0, G1, and G2 glycoforms. The G0 glycoform has no galactose on the terminal GlcNAc residues, and the G1 and G2 glycoforms have one or two terminal galactose residues, respectively, while no N-glycan with terminal sialic acid residue is observed. We show here that under native conditions we can convert the N-glycans of these mAbs to a homogeneous population of G0 glycoform using beta1,4 galactosidase from Streptococcus pneumoniae. The G0 glycoforms of mAbs can be galactosylated with a modified galactose having a chemical handle at the C2 position, such as ketone or azide, using a mutant beta1,4-galactosyltransferase (beta1,4Gal-T1-Y289L). The addition of the modified galactose at a specific glycan residue of a mAb permits the coupling of a biomolecule that carries an orthogonal reactive group. The linking of a biotinylated or a fluorescent dye carrying derivatives selectively occurs with the modified galactose, C2-keto-Gal, at the heavy chain of these mAbs, without altering their antigen binding activities, as shown by indirect enzyme linked immunosorbent assay (ELISA) and fluorescence activated cell sorting (FACS) methods. Our results demonstrate that the linking of cargo molecules to mAbs via glycans could prove to be an invaluable tool for potential drug targeting by immunotherapeutic methods.

Asunto(s)

Anticuerpos Monoclonales/metabolismo , Antígenos de Superficie/análisis , Colorantes Fluorescentes/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Polisacáridos/metabolismo , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Sitios de Unión , Biotinilación , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Galactosa/metabolismo , Glucosamina/metabolismo , Glicosilación , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oligosacáridos/metabolismo , Polisacáridos/química , Receptor ErbB-2/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Coloración y Etiquetado , Especificidad por Sustrato , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

In recent years, various nanotechnology platforms in the area of medical biology, including both diagnostics and therapy, have gained remarkable attention. Moreover, research and development of engineered multifunctional nanoparticles as pharmaceutical drug carriers have spurred exponential growth in applications to medicine in the last decade. Design principles of these nanoparticles, including nanoemulsions, dendrimers, nano-gold, liposomes, drug-carrier conjugates, antibody-drug complexes, and magnetic nanoparticles, are primarily based on unique assemblies of synthetic, natural, or biological components, including but not limited to synthetic polymers, metal ions, oils, and lipids as their building blocks. However, the potential success of these particles in the clinic relies on consideration of important parameters such as nanoparticle fabrication strategies, their physical properties, drug loading efficiencies, drug release potential, and, most importantly, minimum toxicity of the carrier itself. Among these, lipid-based nanoparticles bear the advantage of being the least toxic for in vivo applications, and significant progress has been made in the area of DNA/RNA and drug delivery using lipid-based nanoassemblies. In this review, we will primarily focus on the recent advances and updates on lipid-based nanoparticles for their projected applications in drug delivery. We begin with a review of current activities in the field of liposomes (the so-called honorary nanoparticles), and challenging issues of targeting and triggering will be discussed in detail. We will further describe nanoparticles derived from a novel class of amphipathic lipids called bolaamphiphiles with unique lipid assembly features that have been recently examined as drug/DNA delivery vehicles. Finally, an overview of an emerging novel class of particles (based on lipid components other than phospholipids), solid lipid nanoparticles and nanostructured lipid carriers will be presented. We conclude with a few examples of clinically successful formulations of currently available lipid-based nanoparticles.

Asunto(s)

Lípidos/química , Nanopartículas , Preparaciones Farmacéuticas/administración & dosificación , Animales , ADN/administración & dosificación , Portadores de Fármacos/efectos adversos , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Humanos , Lípidos/efectos adversos , Liposomas , Nanotecnología

RESUMEN

BACKGROUND: Twelve patients with long-standing symptoms of central nervous system (CNS) dysfunction were found to have elevated antibody titres to human herpesvirus-6 (HHV-6) and Epstein-Barr virus (EBV). All patients had four or more of the following neurocognitive symptoms: impaired cognitive functioning, slowed processing speed, sleep disturbance, short-term memory deficit, fatigue and symptoms consistent with depression. OBJECTIVES: We sought to determine whether elevated antibodies to EBV and HHV-6 indicated chronic viral activation in patients with CNS dysfunction and if their symptoms could be improved by suppressing viral activity with oral valganciclovir. STUDY DESIGN: Patients with high IgG antibody titers against HHV-6 and EBV who were suffering from central nervous system dysfunction and debilitating fatigue for more than one year (median 3 years, range 1-8 years) were treated with 6 months of valganciclovir in an open label study. RESULTS: Nine out of 12 (75%) patients experienced near resolution of their symptoms, allowing them all to return to the workforce or full time activites. In the nine patients with a symptomatic response to treatment, EBV VCA IgG titers dropped from 1:2560 to 1:640 (p = 0.008) and HHV-6 IgG titers dropped from a median value of 1:1280 to 1:320 (p = 0.271). Clinically significant hematological toxicity or serious adverse events were not observed among the 12 patients. CONCLUSION: These preliminary clinical and laboratory observations merit additional studies to establish whether this clinical response is mediated by an antiviral effect of the drug, indirectly via immunomodulation or by placebo effect.

Asunto(s)

Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Síndrome de Fatiga Crónica/tratamiento farmacológico , Ganciclovir/análogos & derivados , Infecciones por Roseolovirus/tratamiento farmacológico , Administración Oral , Adulto , Anticuerpos Antivirales/sangre , Antivirales/farmacología , Antivirales/uso terapéutico , Enfermedades del Sistema Nervioso Central/fisiopatología , Enfermedades del Sistema Nervioso Central/virología , Síndrome de Fatiga Crónica/virología , Femenino , Ganciclovir/efectos adversos , Ganciclovir/farmacología , Ganciclovir/uso terapéutico , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 6/efectos de los fármacos , Herpesvirus Humano 6/inmunología , Humanos , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Valganciclovir