RESUMEN
Epidermolysis Bullosa (EB) encompasses a spectrum of mechanobullous disorders caused by rare mutations that result in structural weakening of the skin and mucous membranes. While gene mutated and types of mutations present are broadly predictive of the range of disease to be expected, a remarkable amount of phenotypic variability remains unaccounted for in all but the most deleterious cases. This unexplained variance raises the possibility of genetic modifier effects. We tested this hypothesis using a mouse model that recapitulates a non-Herlitz form of junctional EB (JEB) owing to the hypomorphic jeb allele of laminin gamma 2 (Lamc2). By varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on the strength of dermal-epidermal adhesion and on the clinical severity of JEB in the context of the Lamc2(jeb) mutation. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we demonstrate that Col17a1 is a strong genetic modifier of the non-Herlitz JEB that develops in Lamc2(jeb) mice. This modifier is defined by variations in 1-3 neighboring amino acids in the non-collagenous 4 domain of the collagen XVII protein. These allelic variants alter the strength of dermal-epidermal adhesion in the context of the Lamc2(jeb) mutation and, consequentially, broadly impact the clinical severity of JEB. Overall the results provide an explanation for how normally innocuous allelic variants can act epistatically with a disease causing mutation to impact the severity of a rare, heritable mechanobullous disorder.
Asunto(s)
Autoantígenos/genética , Epidermólisis Ampollosa de la Unión/genética , Epistasis Genética , Laminina/genética , Colágenos no Fibrilares/genética , Animales , Modelos Animales de Enfermedad , Epidermólisis Ampollosa de la Unión/etiología , Epidermólisis Ampollosa de la Unión/patología , Variación Genética , Ratones , Mutación , Colágeno Tipo XVIIRESUMEN
Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using "nunchuck" CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay ("negative Gaussian") membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Transporte Biológico Activo , Membrana Celular/metabolismo , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/genética , Citoesqueleto/metabolismo , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Modelos Biológicos , Modelos Moleculares , Pinocitosis , Liposomas Unilamelares/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
PURPOSE: To image colon-expressed alternatively spliced D domain of tenascin C in preclinical colitis models using near infrared (NIR)-labeled targeted molecular imaging agents. PROCEDURES: A human IgG1 with nanomolar binding affinity specific to the alternatively spliced D domain of tenascin C was generated. Immunohistochemistry identified disease-specific expression of this extracellular matrix protein in the colon of mice given dextran sulfate sodium in the drinking water. The antibody reagent was labeled with the NIR fluorophore IRDye 800CW via amine chemistry and intravenously dosed to evaluate in vivo targeting specificity. Increasing doses of imaging agent were given to estimate the saturating dose. RESULTS: The NIR-labeled proteins successfully targeted colonic lesions in a murine model of colitis. Co-administration of a molar excess competing unlabeled dose reduced normalized uptake in diseased colon by > 70%. Near infrared ex vivo images of colon resected from diseased animals showed saturation at doses exceeding 1 nmol and was confirmed with additional quantitative ex vivo biodistribution. Cellular-level specificity and protein stability were assessed via microscopy. CONCLUSIONS: Our imaging data suggest the alternatively spliced D domain of tenascin C is a promising target for delivery-based applications in inflammatory bowel diseases.
Asunto(s)
Colitis , Enfermedades Inflamatorias del Intestino , Humanos , Animales , Ratones , Tenascina , Distribución Tisular , Colitis/patologíaRESUMEN
The development of nanoscale drug delivery vehicles is an exciting field due to the ability of these vehicles to improve the pharmacokinetic and pharmacodynamic properties of existing therapeutics. These vehicles can improve drug effectiveness and safety by providing benefits such as increased blood circulation, targeted delivery, and controlled release. With regard to the building blocks, amphiphilic polypeptide and polypeptide hybrid (i.e., a macromolecule comprised of a polypeptide and another type of polymer) systems have been recently investigated for their abilities to self-assemble into vesicles. Advances in synthesis methodologies have allowed the development and characterization of many different amphiphilic polypeptide and polypeptide hybrid systems. In this review, we will discuss these vesicle-forming materials in terms of their synthesis, processing, and characterization. In addition, current efforts to use them for drug delivery purposes will be discussed.
Asunto(s)
Materiales Biocompatibles/química , Preparaciones de Acción Retardada/química , Portadores de Fármacos/química , Péptidos/metabolismo , Ingeniería de Proteínas/métodos , Tensoactivos/metabolismo , Animales , Materiales Biocompatibles/metabolismo , Preparaciones de Acción Retardada/metabolismo , Portadores de Fármacos/metabolismo , Composición de Medicamentos , Humanos , Ratones , Microscopía , Péptidos/química , Polimerizacion , Técnicas de Síntesis en Fase Sólida , Tensoactivos/químicaRESUMEN
OBJECTIVES: To assess the ability of monoclonal antibodies (mAbs) specific for fibronectin extra-domain A (FnEDA) to target diseased tissues of mouse collagen induced arthritis (mCIA) models. To explore the parameters of the targeting exhibited by anti-FnEDA mAbs including timing and location. METHODS: Targeting capabilities of anti-FnEDA mAbs were demonstrated by biodistribution study where i.v. injected antibodies were detected by conjugated near-infrared (NIR) fluorophore, 125I label and immunohistochemistry (IHC) of the injected antibody. Location of FnEDA expression in both mCIA and human RA tissue were mapped by IHC. Quantification of anti-FnEDA mAbs targeted to disease tissue was measured by whole-body autoradiography (WBA). Timing of the targeting was interrogated with fluorescent and confocal microscopy using anti-FnEDA mAbs labeled with different fluorophores and injected at different times. RESULTS: Anti-FnEDA mAbs show specific targeting to diseased paws of mCIA animal. The targeting was focused on inflamed synovium which is consistent with FnEDA expression profile in both mCIA and human RA tissues. Anti-FnEDA mAbs accumulated in diseased tissue at pharmacologically relevant concentrations, the targeting was sustained for up to 14 days and FnEDA was able to support targeting of multiple doses of anti-FnEDA mAbs given 5 days apart. CONCLUSION: FnEDA is specifically upregulated in the inflamed tissues of mCIA. Antibodies specific for FnEDA can be useful as molecular delivery vehicles for disease specific targeting of payloads to inflamed joint tissue.
Asunto(s)
Artritis Experimental , Artritis Reumatoide , Animales , Anticuerpos Monoclonales , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Modelos Animales de Enfermedad , Epítopos , Fibronectinas , Humanos , Ratones , Distribución TisularRESUMEN
The block copolypeptide poly(l-homoarginine)(60)-b-poly(l-leucine)(20) (R(60)L(20)) was previously found to self-assemble into versatile vesicles with controllable size and encapsulate hydrophilic cargo. These R(60)L(20) vesicles also demonstrated the ability to cross the cell membrane and transport encapsulated cargo into different cell lines. To assess the potential for using the R(60)L(20) vesicles as drug delivery vehicles further, we have investigated their endocytosis and intracellular trafficking behavior. Using drugs that inhibit different endocytosis pathways, we identified macropinocytosis to be a major process by which the R(60)L(20) vesicles enter HeLa cells. Subsequent immunostaining experiments demonstrated that the vesicles entered the early endosomes but not the lysosomes, suggesting that they recycle back to the cell surface. Overall, our studies indicate that the R(60)L(20) vesicles are able to enter cells intact with their cargos, and although some manage to escape from early endosomes, most are trapped within these intracellular compartments.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Endocitosis/efectos de los fármacos , Endosomas/metabolismo , Homoarginina , Lisosomas/metabolismo , Péptidos/farmacocinética , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/química , Péptidos/farmacologíaRESUMEN
INTRODUCTION: Knowledge that antibodies of the IgG isotype have remarkably extended persistence in circulation and are able to pass through cell barriers has substantial implications. While it is well established that so-called neonatal Fc receptor, FcRn, acts throughout life to confer these unusual properties, its ramifications on clinical medicine and therapeutic uses are not broadly appreciated. SCOPE: Here we discuss basic principles and gaps in understanding of FcRn, including its management of IgG antibodies and along with albumin, its impact on use and design of antibody-based therapeutics, and its genetics.
Asunto(s)
Albúminas/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulina G/inmunología , Complejo Mayor de Histocompatibilidad , Circulación Placentaria/inmunología , Receptores Fc/inmunología , Inmunidad Adaptativa , Albúminas/inmunología , Animales , Autoinmunidad , Circulación Sanguínea/inmunología , Femenino , Humanos , Intestinos/inmunología , Complejo Mayor de Histocompatibilidad/genética , Placenta/inmunología , Embarazo , Ingeniería de ProteínasRESUMEN
Abstract Objectives: To assess the ability of monoclonal antibodies (mAbs) specific for fibronectin extra-domain A (FnEDA) to target diseased tissues of mouse collagen induced arthritis (mCIA) models. To explore the parameters of the targeting exhibited by anti-FnEDA mAbs including timing and location. Methods: Targeting capabilities of anti-FnEDA mAbs were demonstrated by biodistribution study where i.v. injected antibodies were detected by conjugated near-infrared (NIR) fluorophore, 125I label and immunohistochemistry (IHC) of the injected antibody. Location of FnEDA expression in both mCIA and human RA tissue were mapped by IHC. Quantification of anti-FnEDA mAbs targeted to disease tissue was measured by whole-body autoradiography (WBA). Timing of the targeting was interrogated with fluorescent and confocal microscopy using anti-FnEDA mAbs labeled with different fluorophores and injected at different times. Results: Anti-FnEDA mAbs show specific targeting to diseased paws of mCIA animal. The targeting was focused on inflamed synovium which is consistent with FnEDA expression profile in both mCIA and human RA tissues. Anti-FnEDA mAbs accumulated in diseased tissue at pharmacologically relevant concentrations, the targeting was sustained for up to 14 days and FnEDA was able to support targeting of multiple doses of anti-FnEDA mAbs given 5 days apart. Conclusion: FnEDA is specifically upregulated in the inflamed tissues of mCIA. Antibodies specific for FnEDA can be useful as molecular delivery vehicles for disease specific targeting of payloads to inflamed joint tissue.
RESUMEN
An arginine-leucine block copolypeptide (R60 L20 ) is synthesized, which is capable of forming vesicles with controllable sizes, able to transport hydrophilic cargo across the cell membrane, and exhibit relatively low cytotoxicity. The R60 L20 vesicles also possess the ability to deliver DNA into mammalian cells for transfection. Although the transfection efficiency is lower than that of the commercially available transfection agent Lipofectamine 2000, the R60 L20 vesicles are able to achieve transfection with significantly lower cytotoxicity and immunogenicity. This behavior is potentially due to its stronger interaction with DNA which subsequently provides better protection against anionic heparin.
Asunto(s)
Péptidos/farmacología , Transfección/métodos , Liposomas Unilamelares/química , Animales , Aniones , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Células HeLa , Humanos , Interleucina-6/metabolismo , Luz , Lípidos , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Confocal , Plásmidos/metabolismo , Dispersión de RadiaciónRESUMEN
The MHC class I-like Fc receptor (FcRn) is an intracellular trafficking Fc receptor that is uniquely responsible for the extended serum half-life of antibodies of the IgG subclass and their ability to transport across cellular barriers. By performing these functions, FcRn affects numerous facets of antibody biology and pathobiology. Its critical role in controlling IgG pharmacokinetics has been leveraged for the design of therapeutic antibodies and related biologics. FcRn also traffics serum albumin and is responsible for the enhanced pharmacokinetic properties of albumin-conjugated therapeutics. The understanding of FcRn and its therapeutic applications has been limited by a paucity of reliable serological reagents against human FcRn. Here, we describe the properties of a new panel of highly specific monoclonal antibodies (mAbs) directed against human FcRn with diverse epitope specificities. We show that this antibody panel can be used to study the tissue expression pattern of human FcRn, to selectively block IgG and serum albumin binding to human FcRn in vitro and to inhibit FcRn function in vivo. This mAb panel provides a powerful resource for probing the biology of human FcRn and for the evaluation of therapeutic FcRn blockade strategies.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino , Especificidad de Anticuerpos , Antígenos de Histocompatibilidad Clase I/inmunología , Receptores Fc/inmunología , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Ratones , Ratones Noqueados , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Unión Proteica/inmunología , Receptores Fc/antagonistas & inhibidores , Receptores Fc/biosíntesis , Receptores Fc/genética , Albúmina Sérica/inmunología , Albúmina Sérica/metabolismoRESUMEN
Polymeric vesicles are a relatively new class of nanoscale self-assembled materials that show great promise as robust encapsulants. Compared with liposomes, use of polymeric building blocks for membrane formation allows increased stability, stimuli responsiveness and chemical diversity, which may prove advantageous for drug-delivery applications . A major drawback of most polymeric vesicles is the lack of biofunctionality, which restricts their ability to interact with cells and tissues. We have prepared vesicles composed of polyarginine and polyleucine segments that are stable in media, can entrap water soluble species, and can be processed to different sizes and prepared in large quantities. The remarkable feature of these materials is that the polyarginine segments both direct structure for vesicle formation and provide functionality for efficient intracellular delivery of the vesicles. This unique synergy between nanoscale self-assembly and inherent peptide functionality provides a new approach for design of multifunctional materials for drug delivery.