RESUMO
Digital image analysis (DIA) is impacted by the quality of tissue staining. This study examined the influence of preanalytical variables-staining protocol design, reagent quality, section attributes, and instrumentation-on the performance of automated DIA software. Our hypotheses were that (1) staining intensity is impacted by subtle differences in protocol design, reagent quality, and section composition and that (2) identically programmed and loaded stainers will produce equivalent immunohistochemical (IHC) staining. We tested these propositions by using 1 hematoxylin and eosin stainer to process 13 formalin-fixed, paraffin-embedded (FFPE) mouse tissues and by using 3 identically programmed and loaded immunostainers to process 5 FFPE mouse tissues for 4 cell biomarkers. Digital images of stained sections acquired with a commercial whole slide scanner were analyzed by customizable algorithms incorporated into commercially available DIA software. Staining intensity as viewed qualitatively by an observer and/or quantitatively by DIA was affected by staining conditions and tissue attributes. Intrarun and inter-run IHC staining intensities were equivalent for each tissue when processed on a given stainer but varied measurably across stainers. Our data indicate that staining quality must be monitored for each method and stainer to ensure that preanalytical factors do not impact digital pathology data quality.
Assuntos
Biomarcadores Tumorais , Processamento de Imagem Assistida por Computador , Algoritmos , Animais , Imuno-Histoquímica , Camundongos , SoftwareRESUMO
Recent "best practice" recommendations for peripheral nervous system sampling and processing provide guidance regarding nerve preparation for animal toxicity studies. This study explored the impact of delayed fixation, type of fixative, processing cycle times, starting ethanol concentration, and water bath temperature to improve nerve preservation in routinely prepared (paraffin-embedded, hematoxylin and eosin [H&E]-stained) sections. Sciatic nerves from adult Wistar rats (diameter, 1.04 ± 0.1 mm) and young domestic pigs (diameter 5.9 ± 1.2 mm) fixed at necropsy ("0" hours) or 3, 6, 12, or 24 hours after death were immersed in neutral-buffered 10% formalin containing 1.2% methanol (NBF) or methanol-free 4% formaldehyde (MFF) at room temperature. After fixation for 24 hours (rat) or 48 hours (pig), specimens were processed into paraffin, and â¼5-µm-thick sections were flattened on water baths set at 35°C, 40°C, or 45°C before H&E staining. Large-diameter nerves (pig) required longer processing cycles to ensure sufficient paraffin infiltration. For both small-diameter (rat) and large-diameter nerves, structural integrity was optimal if fixation by NBF or MFF occurred within 3 hours and the initial ethanol concentration for tissue processing was lowered to 50%. At all time points, structural preservation of nerve fibers was acceptable using NBF but was better with MFF. Use of a water bath at 35°C reduced processing-related nerve fiber separation within sections.
Assuntos
Nervo Isquiático , Fixação de Tecidos , Testes de Toxicidade , Animais , Fixadores , Formaldeído , Imuno-Histoquímica , Ratos , Ratos Wistar , Manejo de Espécimes , Suínos , ToxicologiaRESUMO
CONTEXT.: The original guideline, "Validating Whole Slide Imaging for Diagnostic Purposes in Pathology," was published in 2013 and included 12 guideline statements. The College of American Pathologists convened an expert panel to update the guideline following standards established by the National Academies of Medicine for developing trustworthy clinical practice guidelines. OBJECTIVE.: To assess evidence published since the release of the original guideline and provide updated recommendations for validating whole slide imaging (WSI) systems used for diagnostic purposes. DESIGN.: An expert panel performed a systematic review of the literature. Frozen sections, anatomic pathology specimens (biopsies, curettings, and resections), and hematopathology cases were included. Cytology cases were excluded. Using the Grading of Recommendations Assessment, Development, and Evaluation approach, the panel reassessed and updated the original guideline recommendations. RESULTS.: Three strong recommendations and 9 good practice statements are offered to assist laboratories with validating WSI digital pathology systems. CONCLUSIONS.: Systematic review of literature following release of the 2013 guideline reaffirms the use of a validation set of at least 60 cases, establishing intraobserver diagnostic concordance between WSI and glass slides and the use of a 2-week washout period between modalities. Although all discordances between WSI and glass slide diagnoses discovered during validation need to be reconciled, laboratories should be particularly concerned if their overall WSI-glass slide concordance is less than 95%.
Assuntos
Interpretação de Imagem Assistida por Computador , Microscopia , Humanos , Biópsia , Interpretação de Imagem Assistida por Computador/métodos , Laboratórios , Microscopia/métodos , Variações Dependentes do Observador , Revisões Sistemáticas como AssuntoRESUMO
Previously we reported that site-specific modification of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) A3C analog with polyethylene glycol (PEG) dramatically improved the pharmacokinetic properties of the protein in rats. However, we could not evaluate the hematological properties of the PEG-A3C protein in rats because human GM-CSF is inactive in rodents. To study the biological effects of PEGylated GM-CSF analogs in rodents we created a homologous site-specific PEGylated murine (mu) GM-CSF (T3C) protein. muGM-CSF and the T3C protein were expressed in Escherichia coli and purified by column chromatography. The purified T3C protein was covalently modified with a linear 20 kDa- or a branched 40 kDa-maleimide-PEG, and the monoPEGylated proteins purified by column chromatography. muGM-CSF, T3C and the two PEG-T3C proteins had comparable in vitro biological activities, as measured by stimulation of proliferation of the murine FDC-P1 cell line. The PEG-T3C proteins had 10- to 25-fold longer circulating half-lives than muGM-CSF and stimulated greater and longer lasting increases in neutrophils and white blood cells than muGM-CSF following a single intravenous or subcutaneous administration to rats. Treatment of rats made neutropenic with cyclophosphamide with the PEG-T3C proteins shortened the time for recovery of neutrophils to normal levels from 9 or 10 days to 5 or 6 days, whereas muGM-CSF showed no benefit versus vehicle solution. Acceleration of neutrophil recovery in cyclophosphamide-treated rats required a minimum of three PEG-T3C treatments over five days. The PEG-T3C proteins should prove useful for evaluating the potential therapeutic benefits of GM-CSF and long-acting GM-CSF proteins in rodent disease models.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacocinética , Hematopoese/efeitos dos fármacos , Polietilenoglicóis/farmacocinética , Animais , Linhagem Celular , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Meia-Vida , Masculino , Neutropenia/tratamento farmacológico , Polietilenoglicóis/administração & dosagem , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacocinéticaRESUMO
With immunohistochemical (IHC) staining increasingly being used to guide clinical decisions, variability in staining quality and reproducibility are becoming essential factors in generating diagnoses using IHC tissue preparations. The current study tested a method to track and quantify the interrun, intrarun, and intersite variability of IHC staining intensity. Our hypothesis was that staining precision between laboratory sites, staining runs, and individual slides may be verified quantitatively, efficiently and effectively utilizing algorithm-based, automated image analysis. To investigate this premise, we tested the consistency of IHC staining in 40 routinely processed (formalin-fixed, paraffin-embedded) human tissues using 10 common antibiomarker antibodies on 2 Dako Omnis instruments at 2 locations (Carpinteria, CA: 30 m above sea level and Longmont, CO: 1500 m above sea level) programmed with identical, default settings and sample pretreatments. Digital images of IHC-labeled sections produced by a whole slide scanner were analyzed by a simple commercially available algorithm and compared with a board-certified veterinary pathologist's semiquantitative scoring of staining intensity. The image analysis output correlated well with pathology scores but had increased sensitivity for discriminating subtle variations and providing reproducible digital quantification across sites as well as within and among staining runs at the same site. Taken together, our data indicate that digital image analysis offers an objective and quantifiable means of verifying IHC staining parameters as a part of laboratory quality assurance systems.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Algoritmos , Análise de Variância , Anticorpos , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Humanos , Fígado/imunologia , Fígado/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Controle de Qualidade , Reprodutibilidade dos Testes , Baço/imunologia , Baço/metabolismo , Coloração e Rotulagem , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/metabolismoRESUMO
The field of digital pathology has rapidly expanded within the last few years with increasing adoption and growth in popularity. As digital pathology matures, it is apparent that we need well-trained individuals to manage our whole-slide imaging systems. This editorial introduces the joint National Society for Histotechnology and Digital Pathology Association online self-paced digital pathology certificate program which was launched in May 2018 that was established to meet this demand. An overview of how this program was developed, the content of the educational modules, and the way that this program is being offered is discussed.
RESUMO
The growth and virulence of Mycobacterium tuberculosis depends on its ability to scavenge host iron, an essential and limited micronutrient in vivo. In this study, we show that ferric iron accumulates both intra- and extra-cellularly in the primary lung lesions of guinea pigs aerosol-infected with the H37Rv strain of M. tuberculosis. Iron accumulated within macrophages at the periphery of the primary granulomatous lesions while extra-cellular ferric iron was concentrated in areas of lesion necrosis. Accumulation of iron within primary lesions was preceded by an increase in expression of heavy chain (H) ferritin, lactoferrin and receptors for transferrin, primarily by macrophages and granulocytes. The increased expression of intra-cellular H ferritin and extra-cellular lactoferrin, more so than transferrin receptor, paralleled the development of necrosis within primary lesions. The deposition of extra-cellular ferric iron within necrotic foci coincided with the accumulation of calcium and phosphorus and other cations in the form of dystrophic calcification. Primary lung lesions from guinea pigs vaccinated with Mycobactrium bovis BCG prior to experimental infection, had reduced iron accumulation as well as H ferritin, lactoferrin and transferrin receptor expression. The amelioration of primary lesion necrosis and dystrophic calcification by BCG vaccination was coincident with the lack of extra-cellular ferric iron and lactoferrin accumulation. These data demonstrate that BCG vaccination ameliorates primary lesion necrosis, dystrophic mineralization and iron accumulation, in part by down-regulating the expression of macrophage H ferritin, lactoferrin and transferrin receptors, in vivo.
Assuntos
Vacina BCG/farmacologia , Calcinose/prevenção & controle , Ferritinas/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Pulmão/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose Pulmonar/metabolismo , Animais , Cálcio/análise , Ferritinas/análise , Cobaias , Imuno-HistoquímicaRESUMO
Recombinant human GH is used to treat GH deficiency in children and adults and wasting in AIDS patients. GH has a circulating half-life of only a few hours in humans and must be administered to patients by daily injection for maximum effectiveness. Previous studies showed that longer-acting forms of GH could be created by modification of GH with multiple 5-kDa amine-reactive polyethylene glycols (PEGs). Eight of nine lysine residues and the N-terminal amino acid were modified to varying extents by amine PEGylation of GH. The amine-PEGylated GH product comprised a complex mixture of multiple PEGylated species that differed from one another in mass, in vitro bioactivity, and in vivo potency. In vitro bioactivity of GH was reduced 100- to 1000-fold by extensive amine PEGylation of the protein. Here we describe a homogeneously modified, mono-PEGylated GH protein that possesses near complete in vitro bioactivity, a long half-life, and increased potency in vivo. The mono-PEGylated GH was created by substituting cysteine for threonine-3 (T3C) of GH, followed by modification of the added cysteine residue with a single 20-kDa cysteine-reactive PEG. The PEG-T3C protein has an approximate 8-fold longer half-life than GH after sc administration to rats. Every other day or every third day administration of PEG-T3C stimulates increases in body weight and tibial epiphysis growth comparable with that produced by daily administration of GH in hypophysectomized rats. Long-acting, mono-PEGylated GH analogs such as PEG-T3C are promising candidates for future testing in humans.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Hormônio do Crescimento Humano/análogos & derivados , Hormônio do Crescimento Humano/farmacocinética , Hipofisectomia , Aumento de Peso/efeitos dos fármacos , Animais , Preparações de Ação Retardada , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Hormônio do Crescimento Humano/química , Masculino , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Ratos , Ratos Sprague-Dawley , Tíbia/efeitos dos fármacos , Resultado do TratamentoRESUMO
OBJECTIVE: Erythropoietin (Epo) bioactivity is significantly reduced by modification of lysine residues with amine-reactive reagents, which are the most commonly used reagents for attaching polyethylene glycols (PEGs) to proteins to improve protein half-life in vivo. The aims of this study were to determine whether Epo bioactivity can be preserved by targeting attachment of maleimide-PEGs to engineered cysteine analogs of Epo, and to determine whether the pegylated Epo cysteine analogs have improved pharmacokinetic properties in vivo. MATERIALS AND METHODS: Thirty-four Epo cysteine analogs were constructed by site-directed mutagenesis and expressed as secreted proteins in baculovirus-infected insect cells. Following purification, monopegylated derivatives of 12 cysteine analogs were prepared using 20-kDa maleimide-PEGs. In vitro biological activities of the proteins were measured in an Epo-dependent cell proliferation assay. Plasma levels of insect cell-expressed wild-type Epo (BV Epo) and a pegylated Epo cysteine analog were quantitated by ELISA following intravenous administration to rats. RESULTS: Biological activities of 17 purified Epo cysteine analogs and 10 purified pegylated Epo cysteine analogs were comparable to that of BV Epo in the in vitro bioassay. The only pegylated cysteine analogs that displayed consistently reduced in vitro bioactivities were substitutions for lysine residues, PEG-K45C and PEG-K154C. The pegylated Epo cysteine analog had a slower initial distribution phase and a longer terminal half-life than BV Epo in rats, but the majority of both proteins were cleared rapidly from the circulation. CONCLUSIONS: Targeted attachment of maleimide-PEGs to engineered Epo cysteine analogs permits rational design of monopegylated Epo analogs with minimal loss of in vitro biological activity. Insect cell-expressed Epo proteins are cleared rapidly from the circulation in rats, possibly due to improper glycosylation. Site-specific pegylation appears to improve the pharmacokinetic properties of Epo.
Assuntos
Substituição de Aminoácidos , Cisteína/química , Eritropoetina/química , Etilenoglicóis/química , Maleimidas/química , Animais , Bioensaio , Linhagem Celular , Cisteína/genética , Eritropoetina/administração & dosagem , Eritropoetina/genética , Eritropoetina/farmacocinética , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas RecombinantesRESUMO
Despite over 60 years of research on antiviral drugs, very few are FDA approved to treat acute viral infections. Rift Valley fever virus (RVFV), an arthropod borne virus that causes hemorrhagic fever in severe cases, currently lacks effective treatments. Existing as obligate intracellular parasites, viruses have evolved to manipulate host cell signaling pathways to meet their replication needs. Specifically, translation modulation is often necessary for viruses to establish infection in their host. Here we demonstrated phosphorylation of p70 S6 kinase, S6 ribosomal protein, and eIF4G following RVFV infection in vitro through western blot analysis and in a mouse model of infection through reverse phase protein microarrays (RPPA). Inhibition of p70 S6 kinase through rapamycin treatment reduced viral titers in vitro and increased survival and mitigated clinical disease in RVFV challenged mice. Additionally, the phosphorylation of p70 S6 kinase was decreased following rapamycin treatment in vivo. Collectively these data demonstrate modulating p70 S6 kinase can be an effective antiviral strategy.
Assuntos
Proteínas Quinases S6 Ribossômicas 70-kDa/efeitos dos fármacos , Vírus da Febre do Vale do Rift/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/antagonistas & inibidores , Animais , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Replicação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Fator de Iniciação Eucariótico 4G/metabolismo , Feminino , Imuno-Histoquímica , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Febre do Vale de Rift/tratamento farmacológico , Febre do Vale de Rift/patologia , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/genética , Vírus da Febre do Vale do Rift/crescimento & desenvolvimento , Vírus da Febre do Vale do Rift/patogenicidade , Sirolimo/metabolismo , Sirolimo/uso terapêutico , Análise de Sobrevida , Células Vero , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
OBJECTIVE: The aim of this study was to determine whether fusion proteins comprising human granulocyte colony-stimulating factor (G-CSF) joined to human immunoglobulin G1 and G4 (IgG1 and IgG4) Fc and C(H) domains are biologically active and have improved pharmacokinetic and hematopoietic properties in vivo. MATERIAL AND METHODS: Chimeric genes encoding human G-CSF fused to the N-termini of the Fc and C(H) domains of human IgG1 and IgG4 were constructed and used to transfect monkey COS cells. The fusion proteins were purified from the conditioned media by protein A affinity chromatography. Bioactivities of the proteins were measured in a G-CSF-dependent in vitro bioassay. Pharmacokinetic and granulopoietic properties of the G-CSF/IgG1-Fc fusion protein were measured in normal rats. RESULTS: The G-CSF/IgG-Fc and G-CSF/IgG-C(H) fusion proteins were secreted from transfected COS cells primarily as disulfide-linked homodimers. On a molar basis, the purified G-CSF/IgG-Fc fusion proteins were as active as G-CSF in in vitro bioassays, whereas bioactivities of the purified G-CSF/IgG-C(H) fusion proteins were decreased 3- to 4-fold. The G-CSF/IgG1-Fc fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating neutrophils and white blood cells than G-CSF following intravenous and subcutaneous administration to rats. CONCLUSION: Fusion of G-CSF to human IgG domains results in homodimeric fusion proteins possessing high in vitro bioactivities, long circulating half-lives, and enhanced hematopoietic properties in vivo.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Hematopoese/efeitos dos fármacos , Imunoglobulina G/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Contagem de Células Sanguíneas , Exame de Medula Óssea , Células COS , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos/genética , Meia-Vida , Humanos , Imunoglobulina G/genética , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Masculino , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Farmacocinética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
Interferon gamma (IFN-γ) is a 28 kDa homodimeric cytokine that exhibits potent immunomodulatory, anti-proliferative, and antiviral properties. The protein is used to treat chronic granulomatous disease and malignant osteopetrosis, and it is under investigation as a treatment for a variety of cancer, fungal and viral diseases. IFN-γ has a short circulating half life in vivo, which necessitates frequent administration to patients. An unusual feature of IFN-γ is that the protein contains no native cysteines. To create a longer-acting and potentially more effective form of the protein, we introduced a cysteine residue into the IFN-γ coding sequence at amino acid position 103, which is located in a surface-exposed, non-helical region of the protein. The added cysteine residue served as the site for targeted modification of the protein with a cysteine-reactive polyethylene glycol (PEG) reagent. The recombinant protein was expressed in bacteria, purified and modified with 10, 20, and 40 kDa maleimide PEGs. The purified, PEGylated proteins had in vitro bioactivities comparable to IFN-γ, as measured using an in vitro cell growth inhibition assay. The PEGylated proteins displayed 20- to 32-fold longer half lives than IFN-γ in rats, and they were significantly more effective than IFN-γ at inhibiting growth of a human tumor xenograft in athymic mice.
Assuntos
Cisteína/química , Inibidores do Crescimento/farmacocinética , Interferon gama/farmacocinética , Polietilenoglicóis/química , Animais , Linhagem Celular Tumoral , Cisteína/genética , Feminino , Inibidores do Crescimento/química , Inibidores do Crescimento/farmacologia , Humanos , Imunomodulação , Interferon gama/química , Interferon gama/farmacologia , Masculino , Camundongos , Camundongos Nus , Mutação/genética , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Previously we showed that granulocyte colony-stimulating factor (G-CSF) in vitro bioactivity is preserved when the protein is joined via a flexible 7 amino acid linker to an immunoglobulin-1 (IgG1)-Fc domain and that the G-CSF/IgG1-Fc fusion protein possessed a longer circulating half-life and improved hematopoietic properties compared to G-CSF in normal rats. We have extended this analysis by comparing the relative hematopoietic potencies of G-CSF/IgG1-Fc to G-CSF in normal mice and to G-CSF and polyethylene glycol (PEG) -modified G-CSF in neutropenic rats. Mice were treated for 5 days using different doses and dosing regimens of G-CSF/IgG1-Fc or G-CSF and circulating neutrophil levels in the animals measured on Day 6. G-CSF/IgG1-Fc stimulated greater increases in blood neutrophils than comparable doses of G-CSF when administered using daily, every other day or every third day dosing regimens. In rats made neutropenic with cyclophosphamide, G-CSF/IgG1-Fc accelerated recovery of blood neutrophils to normal levels (from Day 9 to Day 5) when administered as 5 daily injections or as a single injection on Day 1. By contrast, G-CSF accelerated neutrophil recovery when administered as 5 daily injections, but not when administered as a single injection. G-CSF/IgG1-Fc was as effective as PEG-G-CSF at accelerating neutrophil recovery following a single injection in neutropenic rats. G-CSF/IgG1-Fc and G-CSF/IgG4-Fc fusion proteins in which the 7 amino acid linker was deleted also were effective at accelerating neutrophil recovery following a single injection in neutropenic rats. These studies confirm the enhanced in vivo hematopoietic properties of G-CSF/IgG-Fc fusion proteins.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Hematopoese/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/farmacologia , Neutropenia/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Animais , Contagem de Células Sanguíneas , Modelos Animais de Doenças , Feminino , Expressão Gênica , Ordem dos Genes , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/genética , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Contagem de Leucócitos , Camundongos , Neutropenia/etiologia , Neutrófilos/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Fatores de TempoRESUMO
Interferon beta (IFN-ß) is widely used to ameliorate disease progression in patients with Multiple Sclerosis. IFN-ß has a short half-life in humans, necessitating frequent administration for optimum effectiveness. Covalent modification of IFN-ß with polyethylene glycol (PEG) improves the pharmacokinetic properties of the protein, but can adversely affect the protein's in vitro bioactivity. Random modification of lysine residues in IFN-ß with amine-reactive PEGs decreased the in vitro bioactivity of the protein 50-fold, presumably due to modification of lysine residues near critical receptor binding sites. PEGylated IFN-ß proteins that retained high in vitro bioactivity could be obtained by selective modification of the N-terminus of the protein with PEG. Here we use site-specific PEGylation technology (targeted attachment of a cysteine-reactive-PEG to an engineered cysteine residue in IFN-ß) to identify several additional amino acid positions where PEG can be attached to IFN-ß without appreciable loss of in vitro bioactivity. Unexpectedly, we found that most of the PEG-IFN-ß analogs showed 11- to 78-fold improved in vitro bioactivities relative to their unPEGylated parent proteins and to IFN-ß-1b. In vivo studies showed that a lead PEG-IFN-ß protein had improved pharmacokinetic properties compared to IFN-ß and was significantly more effective than IFN-ß at inhibiting growth of a human tumor xenograft in athymic mice.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Interferon beta/química , Interferon beta/farmacologia , Polietilenoglicóis/química , Substituição de Aminoácidos , Animais , Antineoplásicos/farmacocinética , Proliferação de Células/efeitos dos fármacos , Cisteína/química , Relação Dose-Resposta a Droga , Humanos , Interferon beta-1b , Interferon beta/farmacocinética , Masculino , Camundongos , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Endogenous S-nitrosothiols, including S-nitrosoglutathione (GSNO), mediate nitric oxide (NO)-based signaling, inflammatory responses, and smooth muscle function. Reduced GSNO levels have been implicated in several respiratory diseases, and inhibition of GSNO reductase, (GSNOR) the primary enzyme that metabolizes GSNO, represents a novel approach to treating inflammatory lung diseases. Recently, an association between decreased GSNOR expression and human lung cancer risk was proposed in part based on immunohistochemical staining using a polyclonal GSNOR antibody. GSNOR is an isozyme of the alcohol dehydrogenase (ADH) family, and we demonstrate that the antibody used in those studies cross reacts substantially with other ADH proteins and may not be an appropriate reagent. We evaluated human lung cancer tissue arrays using monoclonal antibodies highly specific for human GSNOR with minimal cross reactivity to other ADH proteins. We verified the presence of GSNOR in ≥85% of specimens examined, and extensive analysis of these samples demonstrated no difference in GSNOR protein expression between cancerous and normal lung tissues. Additionally, GSNOR and other ADH mRNA levels were evaluated quantitatively in lung cancer cDNA arrays by qPCR. Consistent with our immunohistochemical findings, GSNOR mRNA levels were not changed in lung cancer tissues, however the expression levels of other ADH genes were decreased. ADH IB mRNA levels were reduced (>10-fold) in 65% of the lung cancer cDNA specimens. We conclude that the previously reported results showed an incorrect association of GSNOR and human lung cancer risk, and a decrease in ADH IB, rather than GSNOR, correlates with human lung cancer.
Assuntos
Álcool Desidrogenase/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Álcool Desidrogenase/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Anticorpos Monoclonais/metabolismo , Estudos de Casos e Controles , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , RNA Mensageiro/análise , S-Nitrosoglutationa/metabolismoRESUMO
Recombinant interferon alpha-2 (IFN-alpha2) has proven useful for treating a variety of human cancers and viral diseases. IFN-alpha2 has a short circulating half-life in vivo, which necessitates daily or thrice weekly administration to patients. It is possible to extend the circulating half-life of IFN-alpha2 by random modification of lysine residues in the protein with polyethylene glycol (PEG); however, such preparations have heterogeneous structures and low specific activities, and may not provide optimal therapeutic benefits to patients. A long-acting, site-specific, monoPEGylated IFN-alpha2 protein has now been created by targeted attachment of a 20 kDa or a 40 kDa maleimide-PEG to a cysteine analogue of IFN-alpha2, M111C. In vitro bioactivities of the purified 20 kDa and 40 kDa PEG-M111C proteins were within 2- to 3-fold of those of wild type IFN-alpha2 and 7- to 10-fold better than that of a 40 kDa PEG IFN-alpha2 protein created using nontargeted, amine-PEGylation methodology. The 20 kDa and 40 kDa PEG-M111C proteins demonstrated 26- to 38-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. The 20 kDa PEG M111C protein inhibited growth of human NIH:OVCAR-3 cells transplanted into nude mice by >90%, as measured by tumor size, tumor weight, and number of animals with detectable tumors at necropsy, and was significantly more effective than a comparable dose of IFN-alpha2. These data extend our previous findings that bioactivity of IFN-alpha2 can be largely preserved by targeted attachment of PEG moieties to nonessential sites in the protein and provide evidence that site-specific PEGylated IFN-alpha2 proteins possess enhanced tumoricidal properties in vivo.
Assuntos
Antineoplásicos/sangue , Antineoplásicos/farmacologia , Interferon-alfa/sangue , Interferon-alfa/farmacologia , Polietilenoglicóis/química , Animais , Antineoplásicos/farmacocinética , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisteína , Meia-Vida , Humanos , Interferon-alfa/química , Interferon-alfa/farmacocinética , Camundongos , Camundongos Nus , Proteínas Recombinantes/sangue , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologia , Sensibilidade e Especificidade , Transplante HeterólogoRESUMO
Recombinant interferon alpha-2 (IFN-alpha2) is used clinically to treat a variety of viral diseases and cancers. IFN-alpha2 has a short circulating half-life, which necessitates frequent administration to patients. Previous studies showed that it is possible to extend the circulating half-life of IFN-alpha2 by modifying lysine residues of the protein with amine-reactive poly(ethylene glycol) (PEG) reagents. However, amine-PEGylated IFN-alpha2 comprises a heterogeneous product mixture with low specific activity due to the large number and critical locations of lysine residues in IFN-alpha2. In an effort to overcome these problems we determined the feasibility of creating site-specific, mono-PEGylated IFN-alpha2 analogues by introducing a free (unpaired) cysteine residue into the protein, followed by modification of the added cysteine residue with a maleimide-PEG reagent. IFN-alpha2 cysteine analogues were expressed in Escherichia coli and purified, and their in vitro bioactivities were measured in the human Daudi cell line growth inhibition assay. Several cysteine analogues were identified that do not significantly affect in vitro biological activity of IFN-alpha2. Certain of the cysteine analogues, but not wild-type IFN-alpha2, reacted with maleimide-PEG to produce mono-PEGylated proteins. The PEG-Q5C analogue retained high in vitro bioactivity (within 3- to 4-fold of wild-type IFN-alpha2) even when modified with 20- and 40-kDa PEGs. Pharmacokinetic experiments indicated that the 20-kDa PEG-Q5C and 40-kDa PEG-Q5C proteins have 20-fold and 40-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. These studies demonstrate the feasibility of using site-specific PEGylation technology to create a long-acting, mono-PEGylated IFN-alpha2 protein with high specific activity.
Assuntos
Antineoplásicos/farmacologia , Antivirais/farmacologia , Interferon-alfa/farmacologia , Polietilenoglicóis/química , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Cisteína/química , Relação Dose-Resposta a Droga , Escherichia coli/genética , Humanos , Interferon-alfa/química , Lisina/química , Maleimidas/química , Maleimidas/farmacologia , Peso Molecular , Proteínas/química , RatosRESUMO
Granulocyte macrophage colony-stimulating factor (GM-CSF) stimulates proliferation of hematopoietic cells of the macrophage and granulocyte lineages and is used clinically to treat neutropenia and other myeloid disorders. Because of its short circulating half-life, GM-CSF is administered to patients by daily injection. We describe here the engineering of highly potent, long-acting human GM-CSF proteins through site-specific modification of GM-CSF cysteine analogues with a cysteine-reactive poly(ethylene glycol) (PEG) reagent. Thirteen cysteine analogues of GM-CSF were constructed, primarily in nonhelical regions of the protein believed to lie away from the major receptor binding sites. The GM-CSF cysteine analogues were properly processed but insoluble following secretion into the Escherichia coli periplasm. The proteins were refolded and purified by column chromatography. Ten of the cysteine analogues could be modified with a 5-kDa maleimide PEG, and seven of the mono-PEGylated proteins were purified by ion-exchange column chromatography. Biological activities of the 13 cysteine analogues and 7 PEGylated cysteine analogues were comparable to that of wild-type GM-CSF in an in vitro cell proliferation assay using human TF-1 cells. One cysteine analogue was modified with larger 10-, 20-, and 40-kDa PEGs, with only minimal loss of in vitro bioactivity. Pharmacokinetic experiments in rats demonstrated that the PEGylated proteins had up to 47-fold longer circulating half-lives than wild-type GM-CSF. These data demonstrate the utility of site-specific PEGylation for creating highly potent, long-acting GM-CSF analogues and provide further evidence that the nonhelical regions of human GM-CSF examined are largely nonessential for biological activity of the protein.