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Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell receptor signaling and as such is an attractive target for cancer immunotherapy. Although the role of the HPK1 kinase domain (KD) has been extensively characterized, the function of its citron homology domain (CHD) remains elusive. Through a combination of structural, biochemical, and mechanistic studies, we characterize the structure-function of CHD in relationship to KD. Crystallography and hydrogen-deuterium exchange mass spectrometry reveal that CHD adopts a seven-bladed ß-propellor fold that binds to KD. Mutagenesis associated with binding and functional studies show a direct correlation between domain-domain interaction and negative regulation of kinase activity. We further demonstrate that the CHD provides stability to HPK1 protein in cells as well as contributes to the docking of its substrate SLP76. Altogether, this study highlights the importance of the CHD in the direct and indirect regulation of HPK1 function.
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Proteínas Adaptadoras Transductoras de Señales , Proteínas Serina-Treonina Quinasas , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/química , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/química , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , Dominios Proteicos , Cristalografía por Rayos X , Células HEK293RESUMEN
Biotin- and digoxigenin (DIG)-conjugated therapeutic drugs are critical reagents used for the development of anti-drug antibody (ADA) assays for the assessment of immunogenicity. The current practice of generating biotin and DIG conjugates is to label a therapeutic antibody with biotin or DIG via primary amine groups on lysine or N-terminal residues. This approach modifies lysine residues nonselectively, which can impact the ability of an ADA assay to detect those ADAs that recognize epitopes located at or near the modified lysine residue(s). The impact of the lysine modification is considered greater for therapeutic antibodies that have a limited number of lysine residues, such as the variable heavy domain of heavy chain (VHH) antibodies. In this paper, for the first time, we report the application of site-specifically conjugated biotin- and DIG-VHH reagents to clinical ADA assay development using a model molecule, VHHA. The site-specific conjugation of biotin or DIG to VHHA was achieved by using an optimized reductive alkylation approach, which enabled the majority of VHHA molecules labeled with biotin or DIG at the desirable N-terminus, thereby minimizing modification of the protein after labeling and reducing the possibility of missing detection of ADAs. Head-to-head comparison of biophysical characterization data revealed that the site-specific biotin and DIG conjugates demonstrated overall superior quality to biotin- and DIG-VHHA prepared using the conventional amine coupling method, and the performance of the ADA assay developed using site-specific biotin and DIG conjugates met all acceptance criteria. The approach described here can be applied to the production of other therapeutic-protein- or antibody-based critical reagents that are used to support ligand binding assays.
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Biotina , Lisina , Biotina/química , Digoxigenina/química , Anticuerpos , AminasRESUMEN
BACKGROUND: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1]. METHODS: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8+ tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data. RESULTS: ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (KD) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound 89Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing 89Zr immuno-PET reagents. CONCLUSION: 89Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.
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Neoplasias , Tomografía de Emisión de Positrones , Adulto , Humanos , Ratones , Ratas , Animales , Tomografía de Emisión de Positrones/métodos , Indicadores y Reactivos/uso terapéutico , Distribución Tisular , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Inmunoterapia/métodos , Circonio/química , Linfocitos T CD8-positivos/metabolismo , Línea Celular TumoralRESUMEN
Characterization of anti-CD20 antibody binding to CD20 is critical to development of anti-CD20 therapeutics. While SPR is widely used to characterize binding of therapeutics to their targets, its application to the characterization of anti-CD20 therapeutics has been limited by the challenges of obtaining recombinant or native full-length CD20 suitable for ligand binding assays. Extracellular vesicles (EVs) are nanoparticles naturally released from cells that provide a favorable microenvironment for membrane proteins such as CD20 to maintain proper conformation and activity. Here, we report a novel SPR-based assay that enables elucidation of binding kinetics and affinity measurements for anti-CD20 antibody binding to EV-expressed CD20. Our SPR assay is label-free, easy to perform, and demonstrates specific interaction of rituximab and obinutuzumab to CD20 expressed on EVs. The SPR assay revealed that rituximab and obinutuzumab have different binding kinetics and mechanisms to CD20 although both bind to CD20 with high affinity. Our results are consistent with existing literature and verified the validity of this method. The detailed binding kinetics information may also contribute to a better understanding of the interaction between these two antibodies and CD20. Moreover, our method provides a platform with which to characterize other therapeutic antibodies binding to EV-expressed membrane proteins.
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Vesículas Extracelulares , Resonancia por Plasmón de Superficie , Antígenos CD20 , Vesículas Extracelulares/metabolismo , Proteínas de la Membrana , RituximabRESUMEN
The characterization of target binding interactions is critical at each stage of antibody therapeutic development. During early development, it is important to design fit-for-purpose in vitro molecular interaction characterization (MIC) assays that accurately determine the binding kinetics and the affinity of therapeutic antibodies for their targets. Such information enables PK/PD (pharmacokinetics/pharmacodynamics) modeling, estimation of dosing regimens, and assessment of potency. While binding kinetics and affinities seem to be readily obtained, there is little discussion in the literature on how the information should be generated and used in a systematic manner along with other approaches to enable key drug development decisions. The introduction of new antibody modalities poses unique challenges to the development of MIC assays and further increases the need to discuss the impact of developing context-appropriate MIC assays to enable key decision making for these programs. In this paper, we discuss for the first time the challenges encountered when developing MIC assays supporting new antibody modalities. Additionally, through the presentation of several real case studies, we provide strategies to overcome these challenges to enable investigational new drug (IND) filings.
RESUMEN
Immuno-PET is a molecular imaging technique utilizing positron emission tomography (PET) to measure the biodistribution of an antibody species labeled with a radioactive isotope. When applied as a clinical imaging technique, an immuno-PET imaging agent must be manufactured with quality standards appropriate for regulatory approval. This paper describes methods relevant to the chemistry, manufacturing, and controls component of an immuno-PET regulatory filing, such as an investigational new drug application. Namely, the production, quality control, and characterization of the immuno-PET clinical imaging agent, ZED8, an 89Zr-labeled CD8-specific monovalent antibody as well as its desferrioxamine-conjugated precursor, CED8, is described and evaluated. PET imaging data in a human CD8-expressing tumor murine model is presented as a proof of concept that the imaging agent exhibits target specificity and comparable biodistribution across a range of desferrioxamine conjugate loads.
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Anticuerpos Monoclonales/administración & dosificación , Linfocitos T CD8-positivos/inmunología , Leucemia de Células T/diagnóstico por imagen , Imagen Molecular , Tomografía de Emisión de Positrones , Radioisótopos/administración & dosificación , Radiofármacos/administración & dosificación , Circonio/administración & dosificación , Animales , Anticuerpos Monoclonales/química , Línea Celular Tumoral , Femenino , Humanos , Leucemia de Células T/inmunología , Ratones SCID , Valor Predictivo de las Pruebas , Prueba de Estudio Conceptual , Control de Calidad , Radioisótopos/química , Radioisótopos/normas , Radiofármacos/química , Radiofármacos/normas , Circonio/química , Circonio/normasRESUMEN
Binding interactions with the neonatal Fc receptor (FcRn) are one determinant of pharmacokinetic properties of recombinant human monoclonal antibody (rhumAb) therapeutics, and a conserved binding motif in the crystallizable fragment (Fc) region of IgG molecules interacts with FcRn. Surface plasmon resonance (SPR) biosensor assays are often used to characterize interactions between FcRn and rhumAb therapeutics. In such assays, generally either the rhumAb (format 1) or the FcRn protein (format 2) is immobilized on a biosensor chip. However, because evidence suggests that, in some cases, the variable domains of a rhumAb may also affect FcRn binding, we evaluated the effect of SPR assay configuration on binding data. We sought to assess FcRn binding properties of 2 rhumAbs (rhumAb1 and rhumAb2) to FcRn proteins using these 2 biosensor assay formats. The two rhumAbs have greater than 99% sequence identity in the Fc domain but differ in their Fab regions. rhumAb2 contains a positively charged patch in the variable domain that is absent in rhumAb1. Our results showed that binding of rhumAb1 to FcRn was independent of biosensor assay configuration, while binding of rhumAb2 to FcRn was highly SPR assay configuration dependent. Further investigations revealed that the format dependency of rhumAb2-FcRn binding is linked to the basic residues that form a positively charged patch in the variable domain of rhumAb2. Our work highlights the importance of analyzing rhumAb-FcRn binding interactions using 2 alternate SPR biosensor assay configurations. This approach may also provide a simple way to identify the potential for non-Fc-driven FcRn binding interactions in otherwise typical IgGs.
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Anticuerpos Monoclonales , Técnicas Biosensibles/métodos , Antígenos de Histocompatibilidad Clase I , Receptores Fc , Resonancia por Plasmón de Superficie/métodos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/inmunología , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Receptores Fc/química , Receptores Fc/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunologíaRESUMEN
Purification processes for therapeutic antibodies typically exploit multiple and orthogonal chromatography steps in order to remove impurities, such as host-cell proteins. While the majority of host-cell proteins are cleared through purification processes, individual host-cell proteins such as Phospholipase B-like 2 (PLBL2) are more challenging to remove and can persist into the final purification pool even after multiple chromatography steps. With packed-bed chromatography runs using host-cell protein ELISAs and mass spectrometry analysis, we demonstrated that different therapeutic antibodies interact to varying degrees with host-cell proteins in general, and PLBL2 specifically. We then used a high-throughput Protein A chromatography method to further examine the interaction between our antibodies and PLBL2. Our results showed that the co-elution of PLBL2 during Protein A chromatography is highly dependent on the individual antibody and PLBL2 concentration in the chromatographic load. Process parameters such as antibody resin load density and pre-elution wash conditions also influence the levels of PLBL2 in the Protein A eluate. Furthermore, using surface plasmon resonance, we demonstrated that there is a preference for PLBL2 to interact with IgG4 subclass antibodies compared to IgG1 antibodies.
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Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Cromatografía , Lisofosfolipasa/metabolismo , Proteína Estafilocócica A/química , Animales , Células CHO , Cricetinae , Cricetulus , Inmunoglobulina G/metabolismoRESUMEN
Anti-factor D (AFD; FCFD4514S, lampalizumab) is a humanized IgG Fab fragment directed against factor D (fD), a rate-limiting serine protease in the alternative complement pathway (AP). Evaluation of AFD as a potential intravitreal (IVT) therapeutic for dry age-related macular degeneration patients with geographic atrophy (GA) is ongoing. However, it is unclear whether IVT administration of AFD can affect systemic AP activation and potentially compromise host-immune responses. We characterized the pharmacologic properties of AFD and assessed the effects of AFD administered IVT (2 or 20 mg) or intravenous (0.2, 2, or 20 mg) on systemic complement activity in cynomolgus monkeys. For the IVT groups, serum AP activity was reduced for the 20 mg dose group between 2 and 6 hours postinjection. For the intravenous groups, AFD inhibited systemic AP activity for periods of time ranging from 5 minutes (0.2 mg group) to 3 hours (20 mg group). Interestingly, the concentrations of total serum fD increased up to 10-fold relative to predose levels following administration of AFD. Furthermore, AFD was found to inhibit systemic AP activity only when the molar concentration of AFD exceeded that of fD. This occurred in cynomolgus monkeys at serum AFD levels ≥2 µg/ml, a concentration 8-fold greater than the maximum serum concentration observed following a single 10 mg IVT dose in a clinical investigation in patients with GA. Based on these findings, the low levels of serum AFD resulting from IVT administration of a clinically relevant dose are not expected to appreciably affect systemic AP activity.
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Complemento C3a/antagonistas & inhibidores , Factor D del Complemento/antagonistas & inhibidores , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Degeneración Macular/tratamiento farmacológico , Animales , Bovinos , Complemento C3a/inmunología , Factor D del Complemento/inmunología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Inyecciones Intravítreas , Macaca fascicularis , Degeneración Macular/sangre , Degeneración Macular/inmunología , Masculino , Ratones , Resultado del TratamientoRESUMEN
The objectives of this study were to evaluate the relative binding and potencies of three inhibitors of vascular endothelial growth factor A (VEGF), used to treat neovascular age-related macular degeneration, and assess their relevance in the context of clinical outcome. Ranibizumab is a 48 kDa antigen binding fragment, which lacks a fragment crystallizable (Fc) region and is rapidly cleared from systemic circulation. Aflibercept, a 110 kDa fusion protein, and bevacizumab, a 150 kDa monoclonal antibody, each contain an Fc region. Binding affinities were determined using Biacore analysis. Competitive binding by sedimentation velocity analytical ultracentrifugation (SV-AUC) was used to support the binding affinities determined by Biacore of ranibizumab and aflibercept to VEGF. A bovine retinal microvascular endothelial cell (BREC) proliferation assay was used to measure potency. Biacore measurements were format dependent, especially for aflibercept, suggesting that biologically relevant, true affinities of recombinant VEGF (rhVEGF) and its inhibitors are yet to be determined. Despite this assay format dependency, ranibizumab appeared to be a very tight VEGF binder in all three formats. The results are also very comparable to those reported previously.1-3 At equivalent molar ratios, ranibizumab was able to displace aflibercept from preformed aflibercept/VEGF complexes in solution as assessed by SV-AUC, whereas aflibercept was not able to significantly displace ranibizumab from preformed ranibizumab/VEGF complexes. Ranibizumab, aflibercept, and bevacizumab showed dose-dependent inhibition of BREC proliferation induced by 6 ng/mL VEGF, with average IC50 values of 0.088 ± 0.032, 0.090 ± 0.009, and 0.500 ± 0.091 nM, respectively. Similar results were obtained with 3 ng/mL VEGF. In summary Biacore studies and SV-AUC solution studies show that aflibercept does not bind with higher affinity than ranibizumab to VEGF as recently reported,4 and both inhibitors appeared to be equipotent with respect to their ability to inhibit VEGF function.
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Anticuerpos Monoclonales Humanizados/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Bevacizumab , Bovinos , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Unión Proteica , Ranibizumab , Ultracentrifugación , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This "panel-specific" feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.
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Anticuerpos Antiidiotipos , Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales de Origen Murino , Animales , Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Monoclonales de Origen Murino/química , Anticuerpos Monoclonales de Origen Murino/inmunología , Humanos , Hibridomas , Ratones , Ratones Endogámicos BALB CRESUMEN
OBJECTIVE: Numerous observations implicate interferon-α (IFNα) in the pathophysiology of systemic lupus erythematosus (SLE); however, the potential impact of endogenous anti-IFNα autoantibodies (AIAAs) on IFN-pathway and disease activity is unclear. The aim of this study was to characterize IFN-pathway activity and the serologic and clinical profiles of AIAA-positive patients with SLE. METHODS: Sera obtained from patients with SLE (n = 49), patients with rheumatoid arthritis (n = 25), and healthy control subjects (n = 25) were examined for the presence of AIAAs, using a biosensor immunoassay. Serum type I IFN bioactivity and the ability of AIAA-positive sera to neutralize IFNα activity were determined using U937 cells. Levels of IFN-regulated gene expression in peripheral blood were determined by microarray, and serum levels of BAFF, IFN-inducible chemokines, and other autoantibodies were measured using immunoassays. RESULTS: AIAAs were detected in 27% of the serum samples from patients with SLE, using a biosensor immunoassay. Unsupervised hierarchical clustering analysis identified 2 subgroups of patients, IFN(low) and IFN(high) , that differed in the levels of serum type I IFN bioactivity, IFN-regulated gene expression, BAFF, anti-ribosomal P, and anti-chromatin autoantibodies, and in AIAA status. The majority of AIAA-positive patients had significantly lower levels of serum type I IFN bioactivity, reduced downstream IFN-pathway activity, and lower disease activity compared with the IFN(high) patients. AIAA-positive sera were able to effectively neutralize type I IFN activity in vitro. CONCLUSION: Patients with SLE commonly harbor AIAAs. AIAA-positive patients have lower levels of serum type I IFN bioactivity and evidence for reduced downstream IFN-pathway and disease activity. AIAAs may influence the clinical course in SLE by blunting the effects produced by IFNα.
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Autoanticuerpos/inmunología , Interferón-alfa/inmunología , Lupus Eritematoso Sistémico/inmunología , Adulto , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Autoanticuerpos/sangre , Regulación de la Expresión Génica , Humanos , Interferón-alfa/genética , Interferón-alfa/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/metabolismo , Índice de Severidad de la Enfermedad , Transducción de SeñalRESUMEN
Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.
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Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Inmunoglobulina G/química , Animales , Cromatografía por Intercambio Iónico , Cinética , RatasRESUMEN
Cytokines are important cellular signaling proteins involved in inflammation, wound healing and are thought to direct the foreign body response to implanted materials. In this work, polyurethane tubes (25 mm length, 1.02 mm i.d., and 1.65 mm o.d.) were implanted into subcutaneous tissue of male Sprague-Dawley rats. The tubes served as the biomaterial and a means to collect the interstitial fluid that would be exchanged within the tube lumen and the surrounding tissue. After 3 and 7 days, the tubes were explanted and cytokines in the fluid were quantified with a multiplexed cytokine immunoassay. Six cytokines, interleukin-1beta (IL-1beta), IL-4, IL-6, IL-10, macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha), were simultaneously quantified. All cytokine concentrations with the exception of IL-4 and TNF-alpha ranged between low pg/mL to mid ng/mL levels. Neither TNF-alpha nor IL-4 was detected from any sample. These results illustrate the potential of using the tube materials combined with bead-based immunoassays as a direct method for in vivo collection of multiple cytokines in low microliter sample volumes for fixed day biomaterial implant studies.
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Citocinas/metabolismo , Líquido Extracelular/metabolismo , Prótesis e Implantes , Animales , Quimiocina CCL2/análisis , Citocinas/análisis , Líquido Extracelular/química , Líquido Extracelular/inmunología , Estudios de Factibilidad , Inmunoensayo , Mediadores de Inflamación/análisis , Interleucina-10/análisis , Interleucina-1beta/análisis , Interleucina-4/análisis , Interleucina-6/análisis , Masculino , Poliuretanos , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Microdialysis sampling is a method that has promise for collection of important signaling proteins such as cytokines that are involved in every aspect of the immune response. The objective of this study was to determine the role of membrane and tissue alterations on the reduction of interleukin-6 (IL-6) relative recovery of microdialysis probes implanted for 3 and 7 days versus probes implanted on day 0 (acute implant or control probe). Lipopolysaccharide (LPS), a bacterial endotoxin, was used to elicit IL-6 production in the animals. Within the same animal, the recovery of IL-6 through control probes implanted the day of sample collection was compared to the 3- or 7-day implanted probes. Two hours post-LPS administration, the IL-6 concentrations obtained from either the 3-day or 7-day implanted probe were reduced by more than 8-fold when compared to the control probe. The IL-6 concentrations obtained for the 3-day versus control probes 2-h post-LPS injection were 730 +/- 310 and 6440 +/- 1550 pg/mL (mean +/- SD, n = 3), respectively. For the 7-day implant, the IL-6 concentration in the dialysis probe obtained at 2-h post-LPS injection was 990 +/- 590 versus 5520 +/- 1430 pg/mL (mean +/- SD, n = 3) for the control. In vitro recovery experiments and scanning electron microscopy images combined with the in vivo data suggest that the decreased IL-6 content in the dialysate was caused principally by tissue alterations or tissue encapsulation rather than membrane blockage with biological components (membrane biofouling).
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Interleucina-6/aislamiento & purificación , Microdiálisis/métodos , Piel/inmunología , Animales , Membrana Celular/ultraestructura , Lipopolisacáridos/inmunología , Microscopía Electrónica de Rastreo , Ratas , Piel/ultraestructuraRESUMEN
Microdialysis sampling is well-established for sampling small molecules. Recently, there has been an increased interest toward collecting macromolecules using microdialysis sampling. In this work, fluorescein isothiocyanate-labeled dextrans (FITC-dextrans) with molecular weight between 10 and 70 kDa were chosen as representative molecules to study analyte mass transport properties during microdialysis sampling using different lengths (2 and 10 mm) of 100-kDa MWCO polyethersulfone membranes. Experiments were performed in both well-stirred and quiescent phosphate-buffered saline solutions as well as in a 0.3% agar solution. Different fundamental parameters affecting microdialysis sampling of macromolecules, including effective membrane diffusion coefficients, were evaluated. The applicability of the most-often-cited Bungay et al. mass transfer model was compared to experimental data for the FITC-dextrans. For the larger macromolecules, the membrane provides a significant mass transport resistance most likely caused by hindered diffusion. These experimental aspects that are critical to microdialysis sampling of macromolecules are presented.
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Dextranos/química , Microdiálisis/métodos , Dextranos/ultraestructura , Difusión , Fluoresceína-5-Isotiocianato , Microscopía Electrónica de Rastreo , Sensibilidad y Especificidad , SolucionesRESUMEN
Microdialysis sampling probes were used to collect cytokine samples from lipopolysaccharide (LPS)-stimulated macrophages. The probes were immersed into cell culture wells containing either RAW 264.7 or isolated peritoneal macrophages. Dialysates (15 microL) from these wells were subjected to a multiplexed cytokine sandwich immunoassay platform analyzed by flow cytometry that measures up to six separate cytokines, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interferon-gamma (IFN-gamma), macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha) in a single 15-muL sample. In vitro microdialysis sampling relative recovery experiments showed that only IFN-gamma, IL-6, MCP-1, and TNF-alpha could be recovered across a commercially-available 100-kDa MWCO microdialysis membrane. Eleven hours after LPS addition (1 microg/mL), RAW 264.7 macrophages secreted greater than 8000 pg/mL of TNF-alpha and greater than 1000 pg/mL MCP-1. With the peritoneal macrophages, greater than 6000 pg/mL of IL-6, MCP-1, and TNF-alpha were obtained. The maximum dialysate concentrations obtained from the RAW macrophages were 1300 pg/mL for TNF-alpha and 55 pg/mL for MCP-1. Maximum cytokine concentrations from peritoneal macrophage dialysates reached approximately 2000 pg/mL, 1100 pg/mL and 500 pg/mL for TNF-alpha, MCP-1 and IL-6, respectively. Microdialysis sampling allowed for 20-min samples to be collected during the cytokine release from the activated macrophages. These results demonstrate that microdialysis sampling can be used for collection of selected cytokines with improved temporal resolution.