RESUMEN
BACKGROUND: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK-RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. RESULTS: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3ß phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.
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Proteínas Mutantes/metabolismo , Mutación , Osteoclastos/citología , Osteogénesis , Ligando RANK/metabolismo , Animales , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos C57BL , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genética , Osteoclastos/metabolismo , Ligando RANK/genética , Transducción de SeñalRESUMEN
Exome sequencing has identified many candidate genes and mutations for human diseases, but the functional validation of these candidates is a time-consuming and costly process. Here, we describe a method which uses lentiviruses to overexpress calpain mutations that may play a role in dominant diseases such as autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). The use of lentivirus to deliver the mutant calpain allows for a cost-effective, rapid, and efficient approach to test whether or not a candidate gene mutation from exome sequencing acts as the disease-causing allele for a human disorder. This method also provides for a comparison of different candidate mutations from a single gene identified by exome sequencing, as well as elucidating the mechanisms underlying these complex human disorders. Furthermore, this chapter focuses on two different methods to deliver mutant calpain to the cells of the eye, using either a subretinal or an intravitreal injection of the lentivirus into the mouse eye.
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Calpaína/genética , Ojo/metabolismo , Estudios de Asociación Genética/métodos , Proteínas Mutantes/genética , Animales , Calpaína/administración & dosificación , Ojo/patología , Expresión Génica/genética , Humanos , Lentivirus/genética , Proteínas Mutantes/administración & dosificación , Mutación/genética , Retina/metabolismo , Retina/patologíaRESUMEN
Escherichia coli O157:H7 with its traits such as intestinal colonization and fecal-oral route of transmission demands mucosal vaccine development. E. coli secreted protein B (EspB) is one of the key type III secretory system (TTSS) targets for mucosal candidate vaccine due to its indispensable role in the pathogenesis of E. coli O157:H7. However, mucosally administered recombinant proteins have low immunogenicity which could be overcome by the use of mucosal adjuvants. The quest for safe, potent mucosal adjuvant has recognized ΔG fragment of Zonula occludens toxin of Vibrio cholerae with such properties. ΔG enhances mucosal permeability via the paracellular route by altering epithelial tight junction structure in a reversible, ephemeral and non-toxic manner. Therefore, we tested whether recombinant ΔG intranasally co-administered with truncated EspB (EspB + ΔG) could serve as an effective mucosal adjuvant. Results showed that EspB + ΔG group induced higher systemic IgG and mucosal IgA than EspB alone. Moreover, EspB alone developed Th2 type response with IgG1/IgG2a ratio (1.64) and IL-4, IL-10 cytokines whereas that of EspB + ΔG group generated mixed Th1/Th2 type immune response evident from IgG1/IgG2a ratio (1.17) as well as IL-4, IL-10 and IFN-γ cytokine levels compared to control. Sera of EspB + ΔG group inhibited TTSS mediated haemolysis of murine RBCs more effectively compared to EspB, control group and sera of both EspB + ΔG, EspB group resulted in similar levels of efficacious reduction in E. coli O157:H7 adherence to Caco-2 cells compared to control. Moreover, vaccination with EspB + ΔG resulted in significant reduction in E. coli O157:H7 fecal shedding compared to EspB and control group in experimentally challenged streptomycin-treated mice. These results demonstrate mucosal adjuvanticity of ΔG co-administered with EspB in enhancing overall immunogenicity to reduce E. coli O157:H7 shedding.
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Adyuvantes Inmunológicos/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/inmunología , Toxina del Cólera/administración & dosificación , Infecciones por Escherichia coli/prevención & control , Escherichia coli O157/inmunología , Proteínas de Escherichia coli/inmunología , Inmunidad Humoral , Inmunidad Mucosa , Administración Intranasal , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/sangre , Adhesión Bacteriana/efectos de los fármacos , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Derrame de Bacterias , Células CACO-2 , Toxina del Cólera/genética , Transmisión de Enfermedad Infecciosa , Endotoxinas , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/inmunología , Heces/microbiología , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina G/sangre , Ratones Endogámicos BALB C , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Células Th2/inmunologíaRESUMEN
Critical regulation of bile acid (BA) pool size and composition occurs via an intensive molecular crosstalk between the liver and gut, orchestrated by the combined actions of the nuclear Farnesoid X receptor (FXR) and the enterokine fibroblast growth factor 19 (FGF19) with the final aim of reducing hepatic BA synthesis in a negative feedback fashion. Disruption of BA homeostasis with increased hepatic BA toxic levels leads to higher incidence of hepatocellular carcinoma (HCC). While native FGF19 has anti-cholestatic and anti-fibrotic activity in the liver, it retains peculiar pro-tumorigenic actions. Thus, novel analogues have been generated to avoid tumorigenic capacity and maintain BA metabolic action. Here, using BA related Abcb4-/- and Fxr-/- mouse models of spontaneous hepatic fibrosis and HCC, we explored the role of a novel engineered variant of FGF19 protein, called FGF19-M52, which fully retains BA regulatory activity but is devoid of the pro-tumoral activity. Expression of the BA synthesis rate-limiting enzyme Cyp7a1 is reduced in FGF19-M52-treated mice compared to the GFP-treated control group with consequent reduction of BA pool and hepatic concentration. Treatment with the non-tumorigenic FGF19-M52 strongly protects Abcb4-/- and Fxr-/- mice from spontaneous hepatic fibrosis, cellular proliferation and HCC formation in terms of tumor number and size, with significant reduction of biochemical parameters of liver damage and reduced expression of several genes driving the proliferative and inflammatory hepatic scenario. Our data bona fide suggest the therapeutic potential of targeting the FXR-FGF19 axis to reduce hepatic BA synthesis in the control of BA-associated risk of fibrosis and hepatocarcinoma development.
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Ácidos y Sales Biliares/biosíntesis , Productos Biológicos/administración & dosificación , Carcinoma Hepatocelular/prevención & control , Factores de Crecimiento de Fibroblastos/metabolismo , Cirrosis Hepática/prevención & control , Proteínas Mutantes/metabolismo , Animales , Ácidos y Sales Biliares/antagonistas & inhibidores , Modelos Animales de Enfermedad , Factores de Crecimiento de Fibroblastos/administración & dosificación , Factores de Crecimiento de Fibroblastos/genética , Fármacos Gastrointestinales/administración & dosificación , Cirrosis Hepática/complicaciones , Ratones , Ratones Noqueados , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genética , Resultado del TratamientoRESUMEN
The purpose of our study was to determine the toxic effects of hippocampal mutant APP (mAPP) and amyloid beta (Aß) in human mAPP complementary DNA (cDNA) transfected with primary mouse hippocampal neurons (HT22). Hippocampal tissues are the best source of studying learning and memory functions in patients with Alzheimer's disease (AD) and healthy controls. However, investigating immortalized hippocampal neurons that express AD proteins provide an excellent opportunity for drug testing. Using quantitative reverse transcriptase-polymerase chain reaction, immunoblotting & immunofluorescence and transmission electron microscopy, we assessed messenger RNA (mRNA) and protein levels of synaptic, autophagy, mitophagy, mitochondrial dynamics, biogenesis, dendritic protein MAP2 and assessed mitochondrial number and length in mAPP-HT22 cells that express Swedish/Indiana mutations. Mitochondrial function was assessed by measuring the levels of hydrogen peroxide, lipid peroxidation, cytochrome c oxidase activity and mitochondrial adenosine triphosphate. Increased levels of mRNA and protein levels of mitochondrial fission genes, Drp1 and Fis1 and decreased levels fusion (Mfn1, Mfn2 and Opa1) biogenesis (PGC1α, NRF1, NRF2 & TFAM), autophagy (ATG5 & LC3BI, LC3BII), mitophagy (PINK1 & TERT, BCL2 & BNIPBL), synaptic (synaptophysin & PSD95) and dendritic (MAP2) genes were found in mAPP-HT22 cells relative to WT-HT22 cells. Cell survival was significantly reduced mAPP-HT22 cells. GTPase-Drp1 enzymatic activity was increased in mAPP-HT22 cells. Transmission electron microscopy revealed significantly increased mitochondrial numbers and reduced mitochondrial length in mAPP-HT22 cells. These findings suggest that hippocampal accumulation of mAPP and Aß is responsible for abnormal mitochondrial dynamics and defective biogenesis, reduced MAP2, autophagy, mitophagy and synaptic proteins & reduced dendritic spines and mitochondrial structural and functional changes in mAPP hippocampal cells. These observations strongly suggest that accumulation of mAPP and Aß causes mitochondrial, synaptic and autophagy/mitophagy abnormalities in hippocampal neurons, leading to neuronal dysfunction.
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Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Autofagia/genética , Mitofagia/genética , Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/administración & dosificación , Animales , Modelos Animales de Enfermedad , GTP Fosfohidrolasas/genética , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Ratones , Mitocondrias/genética , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genética , Neuronas/efectos de los fármacos , Sinapsis/genética , TransfecciónRESUMEN
In recent years, directed evolution has emerged as an efficient tool to develop and identify novel protein variants. Eimeria tenella microneme-2 (EtMIC2) is a promising vaccine candidate for use against E. tenella infection; however, it only yields partial protection. The present study aimed to improve the immunogenicity and protective efficacy of EtMIC2 through random mutagenesis. Mutagenesis gene libraries of EtMIC2 were generated using error-prone polymerase chain reaction (epPCR), and the corresponding variant proteins were displayed on the yeast cell surface. Variant EtMIC2 proteins with high immunogenicity were screened through fluorescence-activated cell sorting (FACS) based on the affinity between polyclonal antibodies and antigens. Seven effective variant proteins were screened out and heterogeneously expressed in Escherichia coli as subunit vaccines. The protective efficacy of the variant proteins against E. tenella infections was then evaluated in chicken. Two variant proteins (1130 and 2119) displayed higher immunogenicity and protective efficacy than the wild-type EtMIC2 protein against E. tenella infections, increasing body weight gains and significantly decreasing lesion scores and fecal oocyst shedding, and increasing sIgA antibody production and lymphocyte proliferation. These variants displayed potential for use in the development of subunit vaccines for coccidiosis in chickens. The present results also indicate that directed evolution technology is useful for improving the immunogenicity and protective efficacy of parasite antigens.
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Antígenos de Protozoos/inmunología , Coccidiosis/veterinaria , Eimeria tenella/inmunología , Proteínas Mutantes/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/administración & dosificación , Antígenos de Protozoos/genética , Proliferación Celular , Técnicas de Visualización de Superficie Celular , Pollos , Coccidiosis/prevención & control , Eimeria tenella/genética , Escherichia coli/genética , Citometría de Flujo , Inmunoglobulina A Secretora/análisis , Linfocitos/inmunología , Mutagénesis , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Levaduras/genéticaRESUMEN
Wrinkles can have a negative effect on quality of life and Botox is one of the most effective and common treatments. Argireline (Arg0), a mimetic of Botox, has been found to be safer than Botox and effective in reducing wrinkles, with efficacies up to 48% upon 4 weeks of twice daily treatment. However, the skin permeation of Arg0 is poor, due to its large molecular weight and hydrophilicity. Arg0 exists in zwitterionic form and this charged state hindered its skin permeation. Chemical modification of the peptide structure to reduce the formation of zwitterions may result in increased skin permeability. We investigated a total of 4 peptide analogues (Arg0, Arg1, Arg2, Arg3), in terms of skin permeation and wrinkle reduction. The 4 peptides were dissolved in various propylene glycol and water co-solvents. Enhanced human skin permeation was demonstrated by both Arg2 and Arg3 in vitro. On the other hand, the abilities of the 4 analogues to reduce wrinkle formation were also compared using primary human dental pulp stem cells derived neurons. By measuring the inhibition of glutamate release from the neurons in vitro, it was shown that Arg3 was the most effective, followed by Arg1, Arg0 and Arg2.
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Oligopéptidos/administración & dosificación , Oligopéptidos/farmacocinética , Envejecimiento de la Piel/efectos de los fármacos , Adulto , Células Cultivadas , Humanos , Masculino , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genética , Proteínas Mutantes/farmacocinética , Neuronas/efectos de los fármacos , Oligopéptidos/genéticaRESUMEN
A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.
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Proteínas Mutantes/biosíntesis , Neoplasias/tratamiento farmacológico , Proteínas Recombinantes/biosíntesis , alfa-Fetoproteínas/biosíntesis , Línea Celular Tumoral , Fermentación , Humanos , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genética , Pichia/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/genética , alfa-Fetoproteínas/administración & dosificación , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/aislamiento & purificaciónRESUMEN
Enterotoxigenic Escherichia coli (ETEC) strains are a common cause of diarrhea. Extraordinary antigenic diversity has prompted a search for conserved antigens to complement canonical approaches to ETEC vaccine development. EtpA, an immunogenic extracellular ETEC adhesin relatively conserved in the ETEC pathovar, has previously been shown to be a protective antigen following intranasal immunization. These studies were undertaken to explore alternative routes of EtpA vaccination that would permit use of a double mutant (R192G L211A) heat-labile toxin (dmLT) adjuvant. Here, oral vaccination with EtpA adjuvanted with dmLT afforded significant protection against small intestinal colonization, and the degree of protection correlated with fecal IgG, IgA, or total fecal antibody responses to EtpA. Sublingual vaccination yielded compartmentalized mucosal immune responses with significant increases in anti-EtpA fecal IgG and IgA, and mice vaccinated via this route were also protected against colonization. In contrast, while intradermal (i.d.) vaccination achieved high levels of both serum and fecal antibodies against both EtpA and dmLT, mice vaccinated via the i.d. route were not protected against subsequent colonization and the avidity of serum IgG and IgA EtpA-specific antibodies was significantly lower after i.d. immunization compared to other routes. Finally, we demonstrate that antiserum from vaccinated mice significantly impairs binding of LT to cognate GM1 receptors and shows near complete neutralization of toxin delivery by ETEC in vitro Collectively, these data provide further evidence that EtpA could complement future vaccine strategies but also suggest that additional effort will be required to optimize its use as a protective immunogen.
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Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/inmunología , Glicoproteínas de Membrana/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/genética , Administración Oral , Administración Sublingual , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Neutralizantes/análisis , Anticuerpos Neutralizantes/sangre , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/genética , Modelos Animales de Enfermedad , Enterotoxinas/administración & dosificación , Enterotoxinas/genética , Proteínas de Escherichia coli/administración & dosificación , Proteínas de Escherichia coli/genética , Heces/química , Femenino , Inmunoglobulina A/análisis , Inmunoglobulina A/sangre , Inmunoglobulina G/análisis , Inmunoglobulina G/sangre , Inyecciones Intradérmicas , Ratones , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genéticaRESUMEN
Earlier we demonstrated that blocking of interleukin 11 (IL-11) by systemic administration of anti-IL-11 antibodies attenuates severity of Mycobacterium tuberculosis infection in mice. The substitution W147A in the IL-11 molecule creates the form of cytokine capable to disrupt gp130/IL11R signaling complex formation, thus serving as a high-affinity specific antagonist of IL-11-mediated signaling. We hypothesized that this mutant form of IL-11 may serve as an effective tool for inhibition of native IL-11 activity in vivo. We established the recombinant W147A mutant form of IL-11 in an optimized Escherichia coli expression system and administered it as the aerosol in the lungs of M. tuberculosis-susceptible I/St mice infected with M. tuberculosis Our results show that this therapeutic approach markedly inhibits tuberculous inflammation in lungs, increases the survival time of infected animals, and decreases expression of key inflammatory factors at the RNA and protein levels. These findings are a step toward clinical evaluation of the anti-IL-11 therapy for tuberculosis.
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Factores Inmunológicos/administración & dosificación , Interleucina-11/antagonistas & inhibidores , Proteínas Mutantes/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Tuberculosis Pulmonar/tratamiento farmacológico , Administración por Inhalación , Aerosoles , Sustitución de Aminoácidos , Animales , Modelos Animales de Enfermedad , Femenino , Inflamación/patología , Pulmón/patología , Ratones , Análisis de Supervivencia , Usos TerapéuticosRESUMEN
Lipopolysaccharide (LPS)-binding protein (LBP) plays a crucial role in the recognition of bacterial components, such as LPS that causes an immune response. The aim of this study was to compare the different effects of recombinant bovine wild LBP and mutant LBP (67 Ala â Thr) on the LPS-induced inflammatory response of bovine mammary epithelial cells (BMECs). When BMECs were treated with various concentrations of recombinant bovine lipopolysaccharide-binding protein (RBLBP) (1, 5, 10, and 15 µg/mL) for 12 h, RBLBP of 5 µg/mL increased the apoptosis of BMECs induced by LPS without cytotoxicity, and mutant LBP resulted in a higher cell apoptosis than wild LBP did. By gene-chip microarray and bioinformatics, the data identified 2306 differentially expressed genes that were changed significantly between the LPS-induced inflamed BMECs treated with 5 µg/mL of mutant LBP and the BMECs only treated with 10 µg/mL of LPS (fold change ≥2). Meanwhile, 1585 genes were differently expressed between the inflamed BMECs treated with 5 µg/mL of wild LBP and 10 µg/mL of LPS-treated BMECs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that these differentially expressed genes were involved in different pathways that regulate the inflammation response. It predicted that carriers of this mutation increase the risk for a more severe inflammatory response. Our study provides an overview of the gene expression profile between wild LBP and mutant LBP on the LPS-induced inflammatory response of BMECs, which will lead to further understanding of the potential effects of LBP mutations on bovine mammary glands.
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Proteínas de Fase Aguda/administración & dosificación , Proteínas Portadoras/administración & dosificación , Células Epiteliales/efectos de los fármacos , Lipopolisacáridos/química , Glándulas Mamarias Animales/efectos de los fármacos , Glicoproteínas de Membrana/administración & dosificación , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Bovinos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/metabolismo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Biosíntesis de Proteínas/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genéticaRESUMEN
The Endothelial Protein C receptor (EPCR) is essential for the anticoagulant and cytoprotective functions of the Protein C (PC) system. Selected variants of the malaria parasite protein, Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) associated with severe malaria, including cerebral malaria, specifically target EPCR on vascular endothelial cells. Here, we examine the cellular response to PfEMP1 engagement to elucidate its role in malaria pathogenesis. Binding of the CIDRα1.1 domain of PfEMP1 to EPCR obstructed activated PC (APC) binding to EPCR and induced a loss of cellular EPCR functions. CIDRα1.1 severely impaired endothelial PC activation and effectively blocked APC-mediated activation of protease-activated receptor-1 (PAR1) and associated barrier protective effects of APC on endothelial cells. A soluble EPCR variant (E86A-sEPCR) bound CIDRα1.1 with high affinity and did not interfere with (A)PC binding to cellular EPCR. E86A-sEPCR used as a decoy to capture PfEMP1, permitted normal PC activation on endothelial cells, normal barrier protective effects of APC, and greatly reduced cytoadhesion of infected erythrocytes to brain endothelial cells. These data imply important contributions of PfEMP1-induced protein C pathway defects in the pathogenesis of severe malaria. Furthermore, the E86A-sEPCR decoy provides a proof-of-principle strategy for the development of novel adjunct therapies for severe malaria.
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Antígenos CD/metabolismo , Células Endoteliales/fisiología , Malaria/fisiopatología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/metabolismo , Receptores de Superficie Celular/metabolismo , Antígenos CD/genética , Encéfalo/patología , Células Cultivadas , Citoprotección/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/parasitología , Receptor de Proteína C Endotelial , Humanos , Malaria/tratamiento farmacológico , Malaria/parasitología , Proteínas Mutantes/administración & dosificación , Unión Proteica/efectos de los fármacos , Proteína C/metabolismo , Estructura Terciaria de Proteína/genética , Proteínas Protozoarias/genética , Receptor PAR-1/metabolismo , Receptores de Superficie Celular/genética , Transducción de Señal/efectos de los fármacosRESUMEN
The development of novel therapies to treat chronic diabetic ulcers depends upon appropriate animal models for early stage investigation. The NONcNZO10/LtJ mouse is a new polygenic strain developed to more realistically model human metabolic syndrome and obesity-induced type 2 diabetes; however, detailed wound healing properties have not been reported. Herein, we describe a quantitative wound healing study in the NONcNZO10/LtJ mouse using a splinted excisional wound. The rate of wound healing is compared to various controls, and is also quantified in response to topical administration of normal and mutant fibroblast growth factor-1 (FGF-1). Quantitation of reepithelialization shows that the diabetic condition in the NONcNZO10/LtJ mouse is concomitant with a decreased rate of dermal healing. Furthermore, topical administration of a FGF-1/heparin formulation effectively accelerates reepithelialization. A similar acceleration can also be achieved by a stabilized mutant form of FGF-1 formulated in the absence of heparin. Such accelerated rates of healing are not associated with any abnormal histology in the healed wounds. The results identify the NONcNZO10/LtJ mouse as a useful model of impaired wound healing in type 2 diabetes, and further, identify engineered forms of FGF-1 as a potential "second-generation" therapeutic to promote diabetic dermal wound healing.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/complicaciones , Factor 1 de Crecimiento de Fibroblastos/administración & dosificación , Heparina/administración & dosificación , Proteínas Mutantes/administración & dosificación , Úlcera Cutánea/patología , Cicatrización de Heridas/fisiología , Administración Tópica , Animales , Enfermedad Crónica , Diabetes Mellitus Tipo 2/patología , Combinación de Medicamentos , Masculino , Ratones , Repitelización/efectos de los fármacos , Piel/efectos de los fármacos , Piel/patología , Úlcera Cutánea/etiología , Resultado del Tratamiento , Cicatrización de Heridas/efectos de los fármacosRESUMEN
PF-05231023, a long-acting fibroblast growth factor 21 (FGF21) analog, was generated by covalently conjugating two engineered [des-His1, Ala129Cys]FGF21 molecules to a nontargeting human IgG1 κ scaffold. The pharmacokinetics (PK) of PF-05231023 after i.v. and s.c. administration was evaluated in rats and monkeys using two enzyme-linked immunosorbent assays with high specificity for biologically relevant intact N termini (NT) and C termini (CT) of FGF21. Intact CT of FGF21 displayed approximately 5-fold faster systemic plasma clearance (CL), an approximately 2-fold lower steady-state volume of distribution, and at least 5-fold lower bioavailability compared with NT. In vitro serum stability studies in monkeys and humans suggested that the principal CL mechanism for PF-05231023 was degradation by serum proteases. Direct scaling of in vitro serum degradation rates for intact CT of FGF21 underestimated in vivo CL 5-fold, 1.4-fold, and 2-fold in rats, monkeys, and humans, respectively. The reduced steady-state volume of distribution and the bioavailability for intact CT relative to NT in rats and monkeys were compatible with proteolytic degradation occurring outside the plasma compartment via an unidentified mechanism. Human CL and PK profiles for intact NT and CT of FGF21 were well predicted using monkey single-species allometric and Dedrick scaling. Physiologically based pharmacokinetic models incorporating serum stability data and an extravascular extraction term based on differential bioavailability of intact NT and CT of FGF21 in monkeys improved accuracy of human PK predictions relative to Dedrick scaling. Mechanistic physiologically based pharmacokinetic models of this nature may be highly valuable for predicting human PK of fusion proteins, synthetically conjugated proteins, and other complex biologics.
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Anticuerpos Monoclonales Humanizados/farmacología , Drogas en Investigación/farmacocinética , Factores de Crecimiento de Fibroblastos/química , Factores de Crecimiento de Fibroblastos/farmacología , Hipoglucemiantes/farmacocinética , Hipolipemiantes/farmacocinética , Inmunoglobulina G/química , Modelos Biológicos , Proteínas Recombinantes/farmacocinética , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/sangre , Anticuerpos Monoclonales Humanizados/química , Evaluación Preclínica de Medicamentos , Drogas en Investigación/administración & dosificación , Drogas en Investigación/análisis , Drogas en Investigación/química , Factores de Crecimiento de Fibroblastos/administración & dosificación , Factores de Crecimiento de Fibroblastos/sangre , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Semivida , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/sangre , Hipoglucemiantes/química , Hipolipemiantes/administración & dosificación , Hipolipemiantes/sangre , Hipolipemiantes/química , Inmunoglobulina G/sangre , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Cadenas kappa de Inmunoglobulina/sangre , Cadenas kappa de Inmunoglobulina/química , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/metabolismo , Inyecciones Intravenosas , Inyecciones Subcutáneas , Macaca fascicularis , Masculino , Tasa de Depuración Metabólica , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/sangre , Proteínas Mutantes/química , Proteínas Mutantes/farmacocinética , Fragmentos de Péptidos/sangre , Proteolisis , Ratas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/sangre , Proteínas Recombinantes/químicaRESUMEN
BACKGROUND: We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC), which is the most common cause of bacterial diarrhea in children in developing countries and in travelers. METHODS: The vaccine was tested for safety and immunogenicity alone and together with double-mutant heat-labile toxin (dmLT) adjuvant in a double-blind, placebo-controlled Phase I study in 129 Swedish adults. The vaccine consists of four inactivated recombinant E. coli strains overexpressing the major ETEC colonization factors (CFs) CFA/I, CS3, CS5, and CS6 mixed with an LT B-subunit related toxoid, LCTBA. Volunteers received two oral doses of vaccine alone, vaccine plus 10 µg or 25 µg dmLT or placebo. Secretory IgA antibody responses in fecal samples and IgA responses in secretions from circulating intestine-derived antibody secreting cells were assessed as primary measures of vaccine immunogenicity. RESULTS: The vaccine was safe and well tolerated; adverse events were few and generally mild with no significant differences between subjects receiving placebo or vaccine with or without adjuvant. As many as 74% of subjects receiving vaccine alone and 83% receiving vaccine plus 10 µg dmLT showed significant mucosal IgA responses to all five primary vaccine antigens and about 90% of all vaccinees responded to at least four of the antigens. Subjects receiving vaccine plus 10 µg dmLT responded with significantly increased intestine-derived anti-CS6 responses compared to subjects receiving vaccine alone. CONCLUSIONS: The vaccine was safe and broadly immunogenic. dmLT further enhanced mucosal immune responses to CF antigens present in low amounts in the vaccine. Based on these encouraging results, the vaccine will be tested for safety and immunogenicity in different age groups including infants in Bangladesh and for protective efficacy in travelers.
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Adyuvantes Inmunológicos/administración & dosificación , Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/efectos adversos , Vacunas contra Escherichia coli/inmunología , Adyuvantes Inmunológicos/genética , Administración Oral , Adolescente , Adulto , Anticuerpos Antibacterianos/análisis , Células Productoras de Anticuerpos/inmunología , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/genética , Método Doble Ciego , Enterotoxinas/administración & dosificación , Enterotoxinas/genética , Proteínas de Escherichia coli/administración & dosificación , Proteínas de Escherichia coli/genética , Vacunas contra Escherichia coli/administración & dosificación , Heces/química , Femenino , Humanos , Inmunidad Mucosa , Inmunoglobulina A/análisis , Masculino , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genética , Placebos/administración & dosificación , Suecia , Adulto JovenRESUMEN
Studies have contested the innocuousness of Bacillus thuringiensis (Bt) Cry proteins to mammalian cells as well as to mammals microbiota. Thus, this study aimed to evaluate the cytotoxic and antimicrobial effects of two Cry proteins, Cry8Ka5 (a novel mutant protein) and Cry1Ac (a widely distributed protein in GM crops). Evaluation of cyto- and genotoxicity in human lymphocytes was performed as well as hemolytic activity coupled with cellular membrane topography analysis in mammal erythrocytes. Effects of Cry8Ka5 and Cry1Ac upon Artemia sp. nauplii and upon bacteria and yeast growth were assessed. The toxins caused no significant effects on the viability (IC50 > 1,000 µg/mL) or to the cellular DNA integrity of lymphocytes (no effects at 1,000 µg/mL). The Cry8Ka5 and Cry1Ac proteins did not cause severe damage to erythrocytes, neither with hemolysis (IC50 > 1,000 µg/mL) nor with alterations in the membrane. Likewise, the Cry8Ka5 and Cry1Ac proteins presented high LC50 (755.11 and >1,000 µg/mL, resp.) on the brine shrimp lethality assay and showed no growth inhibition of the microorganisms tested (MIC > 1,000 µg/mL). This study contributed with valuable information on the effects of Cry8Ka5 and Cry1Ac proteins on nontarget organisms, which reinforce their potential for safe biotechnological applications.
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Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas Mutantes/genética , Plantas Modificadas Genéticamente/genética , Animales , Artemia/efectos de los fármacos , Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis , Bacterias/efectos de los fármacos , Proteínas Bacterianas/administración & dosificación , Proliferación Celular/efectos de los fármacos , Endotoxinas/administración & dosificación , Eritrocitos/efectos de los fármacos , Proteínas Hemolisinas/administración & dosificación , Humanos , Linfocitos/efectos de los fármacos , Proteínas Mutantes/administración & dosificación , Control Biológico de VectoresRESUMEN
S100A8 is considered proinflammatory by activating TLR4 and/or the receptor for advanced glycation end products. The aim was to investigate inflammatory effects of S100A8 in murine lung. S100A8 was administered to BALB/c mice by nasal inhalation and genes induced over a time-course assessed. LPS was introduced intranasally either alone or 2 h after pretreatment of mice with intranasal application of S100A8 or dexamethasone. A Cys(42)-Ala(42) mutant S100A8 mutant was used to assess whether S100A8's effects were via pathways that were dependent on reactive oxygen species. S100A8 induced IL-10 mRNA, and expression was apparent only in airway epithelial cells. Importantly, it suppressed acute lung injury provoked by LPS inhalation by suppressing mast-cell activation and induction of mediators orchestrating leukocyte recruitment, possibly by reducing NF-κB activation via an IκBα/Akt pathway and by downmodulating pathways generating oxidative stress. The Cys(42)-Ala(42) S100A8 mutant did not induce IL-10 and was less immunosuppressive, indicating modulation by scavenging oxidants. S100A8 inhibition of LPS-mediated injury was as potent, and outcomes were remarkably similar to immunosuppression by dexamethasone. We challenge the notion that S100A8 is an agonist for TLR4 or the receptor for advanced glycation end products. S100A8 induced IL-10 in vivo and initiates a feedback loop that attenuates acute lung injury.
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Lesión Pulmonar Aguda/prevención & control , Calgranulina A/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-10/genética , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/metabolismo , Administración Intranasal , Animales , Antiinflamatorios/administración & dosificación , Western Blotting , Calgranulina A/genética , Análisis por Conglomerados , Dexametasona/administración & dosificación , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Inmunohistoquímica , Interleucina-10/metabolismo , Lipopolisacáridos/administración & dosificación , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
PURPOSE: We successfully developed recombinant human interferon alpha-2b (rhIFN- α 2b) and mutein forms through the site-directed mutagenesis technique. The mutein forms were developed by substituting cysteins at positions 2 and 99 with aspartic acids. The potential adverse effects of these rhIFN- α 2bs were assessed by acute and subchronic studies. METHODS: In the acute study, rhIFN- α 2bs were subcutaneously administered to mice at a single dose of 97.5 µ g/kg, 975 µ g/kg, and 9.75 mg/kg BW and were observed for 14 days. In the subchronic study, single dose of 1.95 µ g/kg and 19.5 µ g/kg, respectively, was given subcutaneously every 3 days for 45 days. RESULTS: No death as well as abnormality in body weight, behavior, presentation of main organs, and value of plasma SGPT and SGOT was observed. Wild type and mutein rhIFN- α 2bs did not show significant adverse effects at dose up to 9.75 mg/kg BW. Administration of these rhIFN- α 2bs given repeatedly did not induce any adverse effect. CONCLUSION: These results suggest that our rhIFN- α 2bs are safe. However, further study is still needed to clarify the safety issue before use in clinical trial.
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Interferón-alfa/efectos adversos , Proteínas Mutantes/efectos adversos , Animales , Conducta Animal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Dosificación Letal Mediana , Ratones , Proteínas Mutantes/administración & dosificación , Especificidad de Órganos/efectos de los fármacos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Coloración y Etiquetado , Tejido Subcutáneo/efectos de los fármacosRESUMEN
There is great interest in the design and development of highly thermostable and immunogenic protein subunit vaccines for biodefense. In this study, we used two orthogonal and complementary computational protein design approaches to generate a series of single-point mutants of RiVax, an attenuated recombinant ricin A chain (RTA) protein subunit vaccine antigen. As assessed by differential scanning calorimetry, the conformational stabilities of the designed mutants ranged from 4°C less stable to 4.5°C more stable than RiVax, depending on solution pH. Two more thermostable (V18P, C171L) and two less thermostable (T13V, S89T) mutants that displayed native-like secondary and tertiary structures (as determined by circular dichroism and fluorescence spectral analysis, respectively) were tested for their capacity to elicit RTA-specific antibodies and toxin-neutralizing activity. Following a prime-boost regimen, we found qualitative differences with respect to specific antibody titers and toxin neutralizing antibody levels induced by the different mutants. Upon a second boost with the more thermostable mutant C171L, a statistically significant increase in RTA-specific antibody titers was observed when compared with RiVax-immunized mice. Notably, the results indicate that single residue changes can be made to the RiVax antigen that increase its thermal stability without adversely impacting the efficacy of the vaccine.
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Mutación Puntual , Ricina/administración & dosificación , Ricina/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Antitoxinas/sangre , Glucemia/análisis , Calorimetría , Dicroismo Circular , Modelos Animales de Enfermedad , Estabilidad de Medicamentos , Ratones , Ratones Endogámicos BALB C , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Intoxicación/prevención & control , Conformación Proteica , Estabilidad Proteica , Ricina/química , Ricina/genética , Ricina/toxicidad , Espectrometría de Fluorescencia , Análisis de Supervivencia , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/química , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/química , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTBA), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant. Two test vaccines were prepared under GMP: (1) A prototype E. coli CFA/I-only formalin-killed whole-cell+LCTBA vaccine, and (2) A "complete" inactivated multivalent ETEC-CF (CFA/I, CS3, CS5 and CS6 antigens) whole-cell+LCTBA vaccine. These vaccines, when given intragastrically alone or together with dmLT in mice, were well tolerated and induced strong intestinal-mucosal IgA antibody responses as well as serum IgG and IgA responses to each of the vaccine CF antigens as well as to LT B subunit (LTB). Both mucosal and serum responses were further enhanced (adjuvanted) when the vaccines were co-administered with dmLT. We conclude that the new multivalent oral ETEC vaccine, both alone and especially in combination with the dmLT adjuvant, shows great promise for further testing in humans.