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1.
Infect Immun ; 88(8)2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32393506

RESUMO

Bacillus anthracis is the causative agent of anthrax disease, presents with high mortality, and has been at the center of bioweapon efforts. The only currently U.S. FDA-approved vaccine to prevent anthrax in humans is anthrax vaccine adsorbed (AVA), which is protective in several animal models and induces neutralizing antibodies against protective antigen (PA), the cell-binding component of anthrax toxin. However, AVA requires a five-course regimen to induce immunity, along with an annual booster, and is composed of undefined culture supernatants from a PA-secreting strain. In addition, it appears to be ineffective against strains that lack anthrax toxin. Here, we investigated a vaccine formulation consisting of recombinant proteins from a surface-localized heme transport system containing near-iron transporter (NEAT) domains and its efficacy as a vaccine for anthrax disease. The cocktail of five NEAT domains was protective against a lethal challenge of inhaled bacillus spores at 3 and 28 weeks after vaccination. The reduction of the formulation to three NEATs (IsdX1, IsdX2, and Bslk) was as effective as a five-NEAT domain cocktail. The adjuvant alum, approved for use in humans, was as protective as Freund's Adjuvant, and protective vaccination correlated with increased anti-NEAT antibody reactivity and reduced bacterial levels in organs. Finally, the passive transfer of anti-NEAT antisera reduced mortality and disease severity, suggesting the protective component is comprised of antibodies. Collectively, these results provide evidence that a vaccine based upon recombinant NEAT proteins should be considered in the development of a next-generation anthrax vaccine.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/prevenção & controle , Anticorpos Antibacterianos/biossíntese , Anticorpos Neutralizantes/biossíntese , Antígenos de Bactérias/imunologia , Bacillus anthracis/efeitos dos fármacos , Administração por Inalação , Compostos de Alúmen/administração & dosagem , Animais , Antraz/imunologia , Antraz/microbiologia , Antraz/mortalidade , Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/genética , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Bacillus anthracis/imunologia , Bacillus anthracis/patogenicidade , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Complemento C5/deficiência , Feminino , Adjuvante de Freund/administração & dosagem , Humanos , Imunogenicidade da Vacina , Camundongos Knockout , Análise de Sobrevida , Vacinação/métodos
2.
Hum Mol Genet ; 27(17): 3079-3098, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29878115

RESUMO

Niemann-Pick type C disease (NP-C) is a fatal neurodegenerative lysosomal storage disorder. It is caused in 95% of cases by a mutation in the NPC1 gene that encodes NPC1, an integral transmembrane protein localized to the limiting membrane of the lysosome. There is no cure for NP-C but there is a disease-modifying drug (miglustat) that slows disease progression but with associated side effects. Here, we demonstrate in a well-characterized mouse model of NP-C that a single administration of AAV-mediated gene therapy to the brain can significantly extend lifespan, improve quality of life, prevent or ameliorate neurodegeneration, reduce biochemical pathology and normalize or improve various indices of motor function. Over-expression of human NPC1 does not cause adverse effects in the brain and correctly localizes to late endosomal/lysosomal compartments. Furthermore, we directly compare gene therapy to licensed miglustat. Even at a low dose, gene therapy has all the benefits of miglustat but without adverse effects. On the basis of these findings and on-going ascendency of the field, we propose intracerebroventricular gene therapy as a potential therapeutic option for clinical use in NP-C.


Assuntos
Adenoviridae/genética , Proteínas de Transporte/administração & dosagem , Modelos Animais de Doenças , Transtornos Neurológicos da Marcha/prevenção & controle , Terapia Genética , Longevidade/genética , Glicoproteínas de Membrana/administração & dosagem , Doença de Niemann-Pick Tipo C/prevenção & controle , Animais , Proteínas de Transporte/fisiologia , Transtornos Neurológicos da Marcha/genética , Transtornos Neurológicos da Marcha/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Inflamação/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Mutação , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia
3.
Nature ; 507(7492): 376-380, 2014 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-24647000

RESUMO

Blood vessel growth in the skeletal system and osteogenesis seem to be coupled, suggesting the existence of molecular crosstalk between endothelial and osteoblastic cells. Understanding the nature of the mechanisms linking angiogenesis and bone formation should be of great relevance for improved fracture healing or prevention of bone mass loss. Here we show that vascular growth in bone involves a specialized, tissue-specific form of angiogenesis. Notch signalling promotes endothelial cell proliferation and vessel growth in postnatal long bone, which is the opposite of the well-established function of Notch and its ligand Dll4 in the endothelium of other organs and tumours. Endothelial-cell-specific and inducible genetic disruption of Notch signalling in mice not only impaired bone vessel morphology and growth, but also led to reduced osteogenesis, shortening of long bones, chondrocyte defects, loss of trabeculae and decreased bone mass. On the basis of a series of genetic experiments, we conclude that skeletal defects in these mutants involved defective angiocrine release of Noggin from endothelial cells, which is positively regulated by Notch. Administration of recombinant Noggin, a secreted antagonist of bone morphogenetic proteins, restored bone growth and mineralization, chondrocyte maturation, the formation of trabeculae and osteoprogenitor numbers in endothelial-cell-specific Notch pathway mutants. These findings establish a molecular framework coupling angiogenesis, angiocrine signals and osteogenesis, which may prove significant for the development of future therapeutic applications.


Assuntos
Osso e Ossos/irrigação sanguínea , Osso e Ossos/metabolismo , Endotélio Vascular/metabolismo , Neovascularização Fisiológica , Osteogênese , Receptores Notch/metabolismo , Animais , Animais Recém-Nascidos , Vasos Sanguíneos/crescimento & desenvolvimento , Desenvolvimento Ósseo/efeitos dos fármacos , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Proliferação de Células , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Endotélio Vascular/citologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteogênese/efeitos dos fármacos , Transdução de Sinais/genética
4.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31036601

RESUMO

Haemophilus ducreyi causes chancroid and is a major cause of cutaneous ulcers in children. Due to environmental reservoirs, both class I and class II H. ducreyi strains persist in cutaneous ulcer regions of endemicity following mass drug administration of azithromycin, suggesting the need for a vaccine. The hemoglobin receptor (HgbA) is a leading vaccine candidate, but its efficacy in animal models is class specific. Controlled human infection models can be used to evaluate vaccines, but only a class I strain (35000HP) has been characterized in this model. As a prelude to evaluating HgbA vaccines in the human model, we tested here whether a derivative of 35000HP containing a class II hgbA allele (FX548) is as virulent as 35000HP in humans. In eight volunteers infected at three sites with each strain, the papule formation rate was 95.8% for 35000HP versus 62.5% for FX548 (P = 0.021). Excluding doses of FX548 that were ≥2-fold higher than those of 35000HP, the pustule formation rate was 25% for 35000HP versus 11.7% for FX548 (P = 0.0053). By Western blot analysis, FX548 and 35000HP expressed equivalent amounts of HgbA in whole-cell lysates and outer membranes. The growth of FX548 and 35000HP was similar in media containing hemoglobin or hemin. By whole-genome sequencing and single-nucleotide polymorphism analysis, FX548 contained no mutations in open reading frames other than hgbA We conclude that by an unknown mechanism, FX548 is partially attenuated in humans and is not a suitable strain for HgbA vaccine efficacy trials in the model.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Cancroide/prevenção & controle , Vacinas Anti-Haemophilus/imunologia , Haemophilus ducreyi/imunologia , Adulto , Alelos , Proteínas de Bactérias/administração & dosagem , Proteínas de Transporte/administração & dosagem , Cancroide/imunologia , Cancroide/microbiologia , Feminino , Vacinas Anti-Haemophilus/administração & dosagem , Vacinas Anti-Haemophilus/genética , Haemophilus ducreyi/genética , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
5.
J Clin Microbiol ; 57(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31068412

RESUMO

Vaccinations with the 10-valent pneumococcal conjugated vaccine (PHiD-CV) started in Iceland in 2011. Protein D (PD) from H. influenzae, which is coded for by the hpd gene, is used as a conjugate in the vaccine and may provide protection against PD-positive H. influenzae We aimed to evaluate the effect of PHiD-CV vaccination on H. influenzae in children, both in carriage and in acute otitis media (AOM). H. influenzae was isolated from nasopharyngeal swabs collected from healthy children attending 15 day care centers in 2009 and from 2012 to 2017 and from middle ear (ME) samples from children with AOM collected from 2012 to 2017. All isolates were identified using PCR for the hpd and fucK genes. Of the 3,600 samples collected from healthy children, 2,465 were culture positive for H. influenzae (68.5% carriage rate); of these, 151 (6.1%) contained hpd-negative isolates. Of the 2,847 ME samples collected, 889 (31.2%) were culture positive for H. influenzae; of these, 71 (8.0%) were hpd negative. Despite the same practice throughout the study, the annual number of ME samples reduced from 660 in 2012 to 330 in 2017. The proportions of hpd-negative isolates in unvaccinated versus vaccinated children were 5.6% and 7.0%, respectively, in healthy carriers, and 5.4% and 7.8%, respectively, in ME samples. The proportion of hpd-negative isolates increased with time in ME samples but not in healthy carriers. The number of ME samples from children with AOM decreased. The PHiD-CV had no effect on the proportion of the hpd gene in H. influenzae from carriage, but there was an increase in hpd-negative H. influenzae in otitis media. The proportions of hpd-negative isolates remained similar in vaccinated and unvaccinated children.


Assuntos
Proteínas de Bactérias/administração & dosagem , Proteínas de Transporte/administração & dosagem , Portador Sadio/microbiologia , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/isolamento & purificação , Imunoglobulina D/administração & dosagem , Lipoproteínas/administração & dosagem , Otite Média/microbiologia , Vacinas Pneumocócicas/administração & dosagem , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Portador Sadio/prevenção & controle , Criança , Pré-Escolar , Orelha Média/microbiologia , Infecções por Haemophilus/prevenção & controle , Haemophilus influenzae/genética , Humanos , Islândia/epidemiologia , Imunoglobulina D/genética , Lactente , Lipoproteínas/genética , Nasofaringe/microbiologia , Otite Média/prevenção & controle , Vacinas Conjugadas/administração & dosagem
6.
J Vasc Surg ; 69(2): 507-515, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30683197

RESUMO

OBJECTIVE: Arteriovenous fistulas created in patients with chronic kidney disease often lose patency and fail to become usable. This prospective trial evaluated the efficacy of vonapanitase, a recombinant human elastase, in promoting radiocephalic fistula patency and use for hemodialysis. METHODS: PATENCY-1 was a double-blind, placebo-controlled trial that enrolled 349 patients on or approaching hemodialysis and being evaluated for radiocephalic arteriovenous fistula creation. Of these, 313 were randomized and 311 treated. Patients were assigned to vonapanitase (n = 210) or placebo (n = 103). The study drug solution was applied topically to the artery and vein for 10 minutes immediately after fistula creation. The primary and secondary end points were primary patency (time to first thrombosis or corrective procedure) and secondary patency (time to abandonment). Tertiary end points included use of the fistula for hemodialysis, fistula maturation by ultrasound, and procedure rates. RESULTS: The Kaplan-Meier estimates of 12-month primary patency were 42% (95% confidence interval [CI], 35-49) and 31% (95% CI, 21-42) for vonapanitase and placebo (P = .25). The Kaplan-Meier estimates of 12-month secondary patency were 74% (95% CI, 68-80) and 61% (95% CI, 51-71) for vonapanitase and placebo (P = .048). The proportions of vonapanitase and placebo patients were 39% and 25% (P = .035) with unassisted use for hemodialysis and 64% and 44% (P = .006) with unassisted plus assisted use. CONCLUSIONS: Vonapanitase treatment did not significantly improve primary patency but was associated with increased secondary patency and use for hemodialysis. Further research is needed to evaluate these end points.


Assuntos
Derivação Arteriovenosa Cirúrgica , Proteínas de Transporte/administração & dosagem , Oclusão de Enxerto Vascular/prevenção & controle , Elastase Pancreática/administração & dosagem , Artéria Radial/cirurgia , Diálise Renal , Trombose/prevenção & controle , Extremidade Superior/irrigação sanguínea , Grau de Desobstrução Vascular/efeitos dos fármacos , Veias/cirurgia , Administração Tópica , Adulto , Idoso , Derivação Arteriovenosa Cirúrgica/efeitos adversos , Proteínas de Transporte/efeitos adversos , Método Duplo-Cego , Feminino , Oclusão de Enxerto Vascular/etiologia , Oclusão de Enxerto Vascular/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Elastase Pancreática/efeitos adversos , Estudos Prospectivos , Artéria Radial/diagnóstico por imagem , Artéria Radial/fisiopatologia , Trombose/etiologia , Trombose/fisiopatologia , Fatores de Tempo , Resultado do Tratamento , Estados Unidos , Veias/diagnóstico por imagem , Veias/fisiopatologia
7.
Biotechnol Bioeng ; 116(1): 155-167, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30229864

RESUMO

Spinal cord injury (SCI) results in paralysis below the injury and strategies are being developed that support axonal regrowth, yet recovery lags, in part, because many axons are not remyelinated. Herein, we investigated strategies to increase myelination of regenerating axons by overexpression of platelet-derived growth factor (PDGF)-AA and noggin either alone or in combination in a mouse SCI model. Noggin and PDGF-AA have been identified as factors that enhance recruitment and differentiation of endogenous progenitors to promote myelination. Lentivirus encoding for these factors was delivered from a multichannel bridge, which we have previously shown creates a permissive environment and supports robust axonal growth through channels. The combination of noggin+PDGF enhanced total myelination of regenerating axons relative to either factor alone, and importantly, enhanced functional recovery relative to the control condition. The increase in myelination was consistent with an increase in oligodendrocyte-derived myelin, which was also associated with a greater density of cells of an oligodendroglial lineage relative to each factor individually and control conditions. These results suggest enhanced myelination of regenerating axons by noggin+PDGF that act on oligodendrocyte-lineage cells post-SCI, which ultimately led to improved functional outcomes.


Assuntos
Proteínas de Transporte/administração & dosagem , Terapia Genética/métodos , Bainha de Mielina/efeitos dos fármacos , Regeneração Nervosa , Fator de Crescimento Derivado de Plaquetas/administração & dosagem , Medicina Regenerativa/métodos , Traumatismos da Medula Espinal/terapia , Animais , Proteínas de Transporte/genética , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Vetores Genéticos , Lentivirus/genética , Camundongos , Fator de Crescimento Derivado de Plaquetas/genética , Resultado do Tratamento
8.
Appl Microbiol Biotechnol ; 103(1): 291-301, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30392121

RESUMO

Craniosynostosis (CS), the premature and pathological fusion of cranial sutures, is a relatively common developmental disorder. Elucidation of the pathways involved and thus therapeutically targeting it would be promising for the prevention of CS. In the present study, we examined the role of BMP pathway in the all-trans retinoic acid (atRA)-induced CS model and tried to target the pathway in vivo via PLGA-based control release. As expected, the posterior frontal suture was found to fuse prematurely in the atRA subcutaneous injection mouse model. Further mechanism study revealed that atRA could repress the proliferation while promote the osteogenic differentiation of suture-derived mesenchymal cells (SMCs). Moreover, BMP signal pathway was found to be activated by atRA, as seen from increased expression of BMPR-2 and pSMAD1/5/9. Recombinant mouse Noggin blocked the atRA-induced enhancement of osteogenesis of SMCs in vitro. In vivo, PLGA microsphere encapsulated with Noggin significantly prevented the atRA-induced suture fusion. Collectively, these data support the hypothesis that BMP signaling is involved in retinoic acid-induced premature fusion of cranial sutures, while PLGA microsphere-based control release of Noggin emerges as a promising strategy for prevention of atRA-induced suture fusion.


Assuntos
Proteínas de Transporte/administração & dosagem , Craniossinostoses/prevenção & controle , Portadores de Fármacos/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/administração & dosagem , Tretinoína/efeitos adversos , Animais , Animais Recém-Nascidos , Materiais Biocompatíveis/administração & dosagem , Materiais Biocompatíveis/química , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proliferação de Células/efeitos dos fármacos , Suturas Cranianas/efeitos dos fármacos , Suturas Cranianas/patologia , Craniossinostoses/etiologia , Modelos Animais de Doenças , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Injeções Subcutâneas , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/patologia , Camundongos Endogâmicos C57BL , Osteogênese/efeitos dos fármacos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Tretinoína/administração & dosagem , Tretinoína/metabolismo
9.
Eur Ann Allergy Clin Immunol ; 51(5): 206-212, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31287265

RESUMO

Summary: Introduction. Sublingual immunotherapy (SLIT) with Pru p 3 can prevent severe allergic reactions to LTP-containing foods, but the standard initiation protocol is time-consuming. Objectives. Establish the safety of a novel ultra-rush initiation protocol for SLIT with Pru p 3. Methods. Prospective study comparing the side effects of the standard vs novel ultra-rush initiation protocols of SLIT with Pru p 3 in patients with anaphylaxis to LTP. Results. Fifteen patients were included (standard initiation, 5; ultra-rush initiation, 10), 80% females. All patients had oropharyngeal pruritus during initiation, 80% with spontaneous recovery, but no other gastro-intestinal, respiratory, cutaneous or systemic side effects occurred in any patient of both groups. Conclusion. The novel ultra-rush protocol halved the build-up time without increasing side effects.


Assuntos
Antígenos de Plantas/administração & dosagem , Antígenos de Plantas/imunologia , Proteínas de Transporte/administração & dosagem , Hipersensibilidade Alimentar/terapia , Proteínas de Plantas/administração & dosagem , Proteínas de Plantas/imunologia , Imunoterapia Sublingual/métodos , Adolescente , Adulto , Anafilaxia/imunologia , Anafilaxia/patologia , Proteínas de Transporte/imunologia , Feminino , Hipersensibilidade Alimentar/imunologia , Humanos , Masculino , Estudos Prospectivos , Prunus persica/imunologia , Prurido/terapia , Estudos Retrospectivos , Imunoterapia Sublingual/efeitos adversos , Adulto Jovem
10.
Am J Respir Cell Mol Biol ; 59(5): 635-647, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29958015

RESUMO

Studies showed that TRIM72 is essential for repair of alveolar cell membrane disruptions, and exogenous recombinant human TRIM72 protein (rhT72) demonstrated tissue-mending properties in animal models of tissue injury. Here we examine the mechanisms of rhT72-mediated lung cell protection in vitro and test the efficacy of inhaled rhT72 in reducing tissue pathology in a mouse model of ventilator-induced lung injury. In vitro lung cell injury was induced by glass beads and stretching. Ventilator-induced lung injury was modeled by injurious ventilation at 30 ml/kg tidal volume. Affinity-purified rhT72 or control proteins were added into culture medium or applied through nebulization. Cellular uptake and in vivo distribution of rhT72 were detected by imaging and immunostaining. Exogenous rhT72 maintains membrane integrity of alveolar epithelial cells subjected to glass bead injury in a dose-dependent manner. Inhaled rhT72 decreases the number of fatally injured alveolar cells, and ameliorates tissue-damaging indicators and cell injury markers after injurious ventilation. Using in vitro stretching assays, we reveal that rhT72 improves both cellular resilience to membrane wounding and membrane repair after injury. Image analysis detected rhT72 uptake by rat alveolar epithelial cells, which can be inhibited by a cholesterol-disrupting agent. In addition, inhaled rhT72 distributes to the distal lungs, where it colocalizes with phosphatidylserine detection on nonpermeabilized lung slices to label wounded cells. In conclusion, our study showed that inhaled rhT72 accumulates in injured lungs and protects lung tissue from ventilator injury, the mechanisms of which include improving cell resilience to membrane wounding, localizing to injured membrane, and augmenting membrane repair.


Assuntos
Proteínas de Transporte/administração & dosagem , Alvéolos Pulmonares/metabolismo , Proteínas Recombinantes/administração & dosagem , Respiração Artificial/efeitos adversos , Lesão Pulmonar Induzida por Ventilação Mecânica/prevenção & controle , Cicatrização , Administração por Inalação , Animais , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Proteínas de Membrana , Camundongos , Alvéolos Pulmonares/lesões , Alvéolos Pulmonares/patologia , Ratos , Lesão Pulmonar Induzida por Ventilação Mecânica/etiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia
11.
Haematologica ; 103(12): 2097-2108, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30026342

RESUMO

Ibrutinib and acalabrutinib are irreversible inhibitors of Bruton tyrosine kinase used in the treatment of B-cell malignancies. They bind irreversibly to cysteine 481 of Bruton tyrosine kinase, blocking autophosphorylation on tyrosine 223 and phosphorylation of downstream substrates including phospholipase C-γ2. In the present study, we demonstrate that concentrations of ibrutinib and acalabrutinib that block Bruton tyrosine kinase activity, as shown by loss of phosphorylation at tyrosine 223 and phospholipase C-γ2, delay but do not block aggregation in response to a maximally-effective concentration of collagen-related peptide or collagen. In contrast, 10- to 20-fold higher concentrations of ibrutinib or acalabrutinib block platelet aggregation in response to glycoprotein VI agonists. Ex vivo studies on patients treated with ibrutinib, but not acalabrutinib, showed a reduction of platelet aggregation in response to collagen-related peptide indicating that the clinical dose of ibrutinib but not acalabrutinib is supramaximal for Bruton tyrosine kinase blockade. Unexpectedly, low concentrations of ibrutinib inhibited aggregation in response to collagen-related peptide in patients deficient in Bruton tyrosine kinase. The increased bleeding seen with ibrutinib over acalabrutinib is due to off-target actions of ibrutinib that occur because of unfavorable pharmacodynamics.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Agamaglobulinemia/tratamento farmacológico , Plaquetas/efeitos dos fármacos , Doenças Genéticas Ligadas ao Cromossomo X/tratamento farmacológico , Glicoproteínas da Membrana de Plaquetas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/genética , Tirosina Quinase da Agamaglobulinemia/metabolismo , Agamaglobulinemia/sangue , Agamaglobulinemia/genética , Benzamidas/administração & dosagem , Benzamidas/metabolismo , Plaquetas/metabolismo , Proteínas de Transporte/administração & dosagem , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Mutação , Peptídeos/administração & dosagem , Piperidinas , Ativação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , Glicoproteínas da Membrana de Plaquetas/agonistas , Inibidores de Proteínas Quinases/metabolismo , Pirazinas/administração & dosagem , Pirazinas/metabolismo , Pirazóis/administração & dosagem , Pirazóis/metabolismo , Pirimidinas/administração & dosagem , Pirimidinas/metabolismo
12.
Mol Pharm ; 15(12): 5781-5792, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30392378

RESUMO

Polyglutamine diseases are a set of progressive neurodegenerative disorders caused by misfolding and aggregation of mutant CAG RNA and polyglutamin protein. To date, there is a lack of effective therapeutics that can counteract the polyglutamine neurotoxicity. Two peptidylic inhibitors, QBP1 and P3, targeting the protein and RNA toxicities, respectively, have been previously demonstrated by us with combinational therapeutic effects on the Drosophila polyglutamine disease model. However, their therapeutic efficacy has never been investigated in vivo in mammals. The current study aims to (a) develop a brain-targeting delivery system for both QBP1 and L1P3V8 (a lipidated variant of P3 with improved stability) and (b) evaluate their therapeutic effects on the R6/2 transgenic mouse model of polyglutamine disease. Compared with intravenous administration, intranasal administration of QBP1 significantly increased its brain-to-plasma ratio. In addition, employment of a chitosan-containing in situ gel for the intranasal administration of QBP1 notably improved its brain concentration for up to 10-fold. Further study on intranasal cotreatment with the optimized formulation of QBP1 and L1P3V8 in mice found no interference on the brain uptake of each other. Subsequent efficacy evaluation of 4-week daily QBP1 (16 µmol/kg) and L1P3V8 (6 µmol/kg) intranasal cotreatment in the R6/2 mice demonstrated a significant improvement on the motor coordination and explorative behavior of the disease mice, together with a full suppression on the RNA- and protein-toxicity markers in their brains. In summary, the current study developed an efficient intranasal cotreatment of the two peptidylic inhibitors, QBP1 and L1P3V8, for their brain-targeting, and such a novel therapeutic strategy was found to be effective on a transgenic polyglutamine disease mouse model.


Assuntos
Proteínas de Transporte/administração & dosagem , Transtornos Heredodegenerativos do Sistema Nervoso/tratamento farmacológico , Oligopeptídeos/administração & dosagem , Peptídeos/administração & dosagem , Peptídeos/metabolismo , RNA Mensageiro/antagonistas & inibidores , Administração Intranasal , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Proteínas de Transporte/farmacocinética , Modelos Animais de Doenças , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada/métodos , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligopeptídeos/farmacocinética , Peptídeos/farmacocinética , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Expansão das Repetições de Trinucleotídeos/genética
13.
Fish Shellfish Immunol ; 76: 247-259, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29518559

RESUMO

Recently, several immunostimulants such as ß-glucan, microbial and plant products have been used as dietary supplements to combat disease outbreaks in aquaculture. The present study investigates the potential of Portunus pelagicus ß-1, 3 glucan binding protein based zinc oxide nanoparticles (Ppß-GBP-ZnO NPs) supplemented diet on growth, immune response and disease resistance in Mozambique tilapia, Oreochromis mossambicus. The immune-related protein ß-GBP was purified from the haemolymph of P. pelagicus using Sephadex G-100 affinity column chromatography. Ppß-GBP-ZnO NPs was physico- chemically characterized and experimental feed was formulated. Fish were separately fed with commercial diet (control-group I) and Ppß-GBP (group II, III, IV), Ppß-GBP-ZnO NPs (group V, VI, VII), chem-ZnO NPs (VIII, IX, X) mixed diet at the concentration of 0.001%, 0.002% and 0.004% respectively. Triplicate groups of O. mossambicus were fed with experimental diets twice a day for 30 days. Fish receiving Ppß-GBP-ZnO NPs supplemented diet showed a significant increase (P < 0.05) in growth performance. Cellular immune responses (myeloperoxidase activity, lysozyme activity and reactive oxygen species activity) and humoral immune responses (complement activity, antiprotease activity and alkaline phosphatase activity) were evaluated at an interval of 15 days during the feeding trial. Results demonstrate that both cellular and humoral immune responses were substantially increased (P < 0.05) in fish fed with 0.004% of Ppß-GBP-ZnO NPs supplemented diet than others. Antibiofilm potential of Ppß-GBP-ZnO NPs against Aeromonas hydrophila was visualized through confocal laser scanning microscopy (CLSM), which reveals reduction in the preformed biofilm thickness to 10 µm  at the concentration of 50 µg/ml. Furthermore, after 30 days of feeding trial, fish were challenged with aquatic fish pathogen A. hydrophila (1 × 107 cells ml-1) through intraperitoneal injection. Challenge study displayed a reduced mortality rate in fish fed with diet containing Ppß-GBP-ZnO NPs. Thus our study suggests that dietary supplementation of Ppß-GBP-ZnO NPs at 0.004% may have a potential effect to enhance the immune system and survival of O. mossambicus.


Assuntos
Proteínas de Transporte/metabolismo , Resistência à Doença/efeitos dos fármacos , Doenças dos Peixes/imunologia , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Lectinas/metabolismo , Tilápia/imunologia , Óxido de Zinco/farmacologia , Aeromonas hydrophila/imunologia , Ração Animal/análise , Animais , Braquiúros/química , Proteínas de Transporte/administração & dosagem , Dieta/veterinária , Suplementos Nutricionais/análise , Feminino , Infecções por Bactérias Gram-Negativas/imunologia , Lectinas/administração & dosagem , Masculino , Nanopartículas Metálicas/administração & dosagem , Distribuição Aleatória , Tilápia/crescimento & desenvolvimento , Óxido de Zinco/administração & dosagem , Óxido de Zinco/metabolismo
14.
Biol Pharm Bull ; 41(4): 546-554, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29607927

RESUMO

To overcome the difficulty in delivery of biopharmaceuticals such as peptides and proteins to the brain, several approaches combining the ligands and antibodies targeting the blood-brain barrier (BBB) have been tried. However, these are inefficient in terms of their permeability through the BBB and structural modification of bioactive drugs. In the present study, we therefore examined the usefulness of a noncovalent method using the cell-penetrating peptides (CPPs) such as octaarginine (R8) as a suitable brain delivery strategy for biopharmaceuticals. A safety examination using microvascular endothelial model bEnd.3 cells clarified that R8 was the safest among the CPPs tested in this study. The cellular uptake study demonstrated that coincubation with R8 enhanced the uptake of model peptide drug insulin by bEnd.3 cells in a concentration-dependent and a temperature-independent manner. Furthermore, an in vivo study with rats showed that the accumulation of insulin in the deeper region of the brain, i.e., hippocampus, significantly increased after the intravenous coadministration of insulin with D-R8 without altering the insulin disposition in plasma. Thus, the present study provided the first evidence suggesting that the noncovalent method with CPPs is one of the strategic options for brain delivery of biopharmaceuticals via intravenous injection.


Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/administração & dosagem , Peptídeos Penetradores de Células/administração & dosagem , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Oligopeptídeos/administração & dosagem , Animais , Transporte Biológico , Proteínas de Transporte/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacocinética , Produtos do Gene tat/química , Hipoglicemiantes/sangue , Hipoglicemiantes/farmacocinética , Insulina/sangue , Insulina/farmacocinética , Masculino , Camundongos , Oligopeptídeos/farmacocinética , Ratos Sprague-Dawley , Distribuição Tecidual
15.
Biol Pharm Bull ; 41(2): 239-246, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29386483

RESUMO

This present study aimed to determine the optimal oral insulin delivery conditions that would maximize the utility of cell-penetrating peptides (CPPs) by using a noncovalent strategy. We first compared the effectiveness of two potential CPPs, penetratin and its analog PenetraMax, as absorption enhancers for insulin. The combined effect was evaluated under in vivo oral administration conditions. Both D-forms of CPPs were highly effective for increasing the oral absorption of insulin, and D-PenetraMax showed a more rapid onset of absorption enhancement effects compared with those of D-penetratin. However, synergistic absorption enhancement effects after combination treatment were not observed. Next, we tried a theoretical approach to establish optimized oral insulin delivery conditions. A surface plasmon resonance (SPR)-based analysis demonstrated that binding between insulin and penetratin (2 mM) might be saturated at 100-500 µM penetratin, while the bound concentration of penetratin could increase in accordance with an increased concentration of mixed insulin. To test this hypothesis, we investigated the effectiveness of different insulin doses in the gastric pH-neutralized mice. The results showed that the dissociation of noncovalent complexes of insulin and CPPs at the low gastric pH was prevented in these mice. Our findings clearly suggested that a noncovalent strategy with CPPs represents an effective approach for the L-form of CPP to increase the concentration of CPP-bound insulin to attain greater absorption of insulin, although this approach may not be appropriate for the D-form of CPP. Our findings will contribute to the development of oral dosage forms of insulin for noncovalent strategies involving CPP.


Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Sistemas de Liberação de Medicamentos , Hipoglicemiantes/administração & dosagem , Insulina Regular Humana/administração & dosagem , Absorção Intestinal/efeitos dos fármacos , Administração Oral , Animais , Animais não Endogâmicos , Disponibilidade Biológica , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/química , Proteínas de Transporte/farmacocinética , Proteínas de Transporte/farmacologia , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacocinética , Peptídeos Penetradores de Células/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Famotidina/farmacologia , Ácido Gástrico/química , Ácido Gástrico/metabolismo , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Antagonistas dos Receptores H2 da Histamina/farmacologia , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Insulina Regular Humana/genética , Insulina Regular Humana/farmacocinética , Insulina Regular Humana/farmacologia , Ligantes , Masculino , Camundongos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologia , Estereoisomerismo , Ressonância de Plasmônio de Superfície
16.
Allergol Immunopathol (Madr) ; 46(5): 482-490, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29786518

RESUMO

BACKGROUND: Lipid transfer protein (LTP) is a major fruit allergen. It has, however, recently been revealed that the systemic reaction in peach-allergic patients is related not only to LTP (Pru p 3) but also to gibberellin-regulated protein (Pru p 7). We investigated recombinant Pru p 7 (rPru p 7) for its potential use in worldwide standardization for the diagnosis of peach allergy. METHODS: Natural Pru p 7 (nPru p 7) was purified from peach crude extract using a monoclonal antibody affinity column. Complementary DNA for Pru p 7 was cloned and expressed in Escherichia coli and Pichia pastoris. Serum immunoglobulin (Ig) E in peach-allergic patients was examined by enzyme-linked immunosorbent assay (ELISA) using nPru p 7 and rPru p 7 (E. coli product: erPru p 7 and P. pastoris product: prPru p 7). RESULTS: Peach-allergic patients (n=27) were diagnosed and categorized into oral reaction (n=10) or systemic reaction (n=17). The nPru p 7 positivity based on serum IgE levels was 52% in the systemic-reaction group and 0% in the oral-reaction group (P<0.05). In the systemic-reaction group, there was no significant difference in reactivity between nPru p 7 and prPru p 7, but the reactivity of erPru p 7 was significantly lower than those of nPru p 7 and prPru p 7 (P<0.05). CONCLUSIONS: We found that prPru p 7 exhibited reactivity in ELISA comparable to that of nPru p 7 for the diagnosis of peach allergy with systemic reaction.


Assuntos
Antígenos de Plantas/imunologia , Hipersensibilidade Alimentar/diagnóstico , Imunoglobulina E/sangue , Prunus persica/efeitos adversos , Adolescente , Adulto , Antígenos de Plantas/administração & dosagem , Antígenos de Plantas/efeitos adversos , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/efeitos adversos , Proteínas de Transporte/imunologia , Criança , Ensaio de Imunoadsorção Enzimática , Feminino , Hipersensibilidade Alimentar/sangue , Hipersensibilidade Alimentar/imunologia , Giberelinas/administração & dosagem , Giberelinas/efeitos adversos , Giberelinas/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/imunologia , Adulto Jovem
17.
Mol Pharm ; 14(3): 916-927, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28094952

RESUMO

Insulin is now considered to be a new drug candidate for treating dementias, such as Alzheimer's disease, whose pathologies are linked to insulin resistance in the brain. Our recent work has clarified that a noncovalent strategy involving cell-penetrating peptides (CPPs) can increase the direct transport of insulin from the nasal cavity into the brain parenchyma. The present study aimed to determine whether the brain insulin level increased by intranasal coadministration of insulin with the CPP penetratin has potential for treating dementia. The pharmacological actions of insulin were investigated at different stages of memory impairment using a senescence-accelerated mouse-prone 8 (SAMP8) model. The results of spatial learning tests suggested that chronic intranasal administration of insulin with l-penetratin to SAMP8 slowed the progression of memory loss in the early stage of memory impairment. However, contrary to expectations, this strategy using penetratin was ineffective in recovering the severe cognitive dysfunction in the progressive stage, which involves brain accumulation of amyloid ß (Aß). Immunohistological examination of hippocampal regions of samples from SAMP8 in the progressive stage suggested that accelerated nose-to-brain insulin delivery had a partial neuroprotective function but unexpectedly increased Aß plaque deposition in the hippocampus. These findings suggest that the efficient nose-to-brain delivery of insulin combined with noncovalent CPP strategy has different effects on dementia during the mild and progressive stages of cognitive dysfunction.


Assuntos
Envelhecimento/efeitos dos fármacos , Demência/tratamento farmacológico , Hipocampo/metabolismo , Insulina/administração & dosagem , Transtornos da Memória/tratamento farmacológico , Mucosa Nasal/metabolismo , Administração Intranasal/métodos , Peptídeos beta-Amiloides/metabolismo , Animais , Proteínas de Transporte/administração & dosagem , Peptídeos Penetradores de Células/metabolismo , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/metabolismo , Demência/metabolismo , Modelos Animais de Doenças , Masculino , Transtornos da Memória/metabolismo , Camundongos , Placa Amiloide/metabolismo
18.
Biochem Biophys Res Commun ; 476(4): 654-664, 2016 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-27240954

RESUMO

NS4B of hepatitis C virus (HCV) initiates membrane web formation, binds RNA and other HCV proteins for viral replication complex (RC) formation, hydrolyses NTP, and inhibits innate anti-viral immunity. Thus, NS4B is an attractive target of a novel anti-HCV agent. In this study, humanized-nanobodies (VHs/VHHs) that bound to recombinant NS4B were produced by means of phage display technology. The nanobodies were linked molecularly to a cell penetrating peptide, penetratin (PEN), for making them cell penetrable (become transbodies). Human hepatic (Huh7) cells transfected with HCV JFH1-RNA that were treated with transbodies from four Escherichia coli clones (PEN-VHH7, PEN-VHH9, PEN-VH33, and PEN-VH43) had significant reduction of HCV RNA amounts in their culture fluids and intracellularly when compared to the transfected cells treated with control transbody and medium alone. The results were supported by the HCV foci assay. The transbody treated-transfected cells also had upregulation of the studied innate cytokine genes, IRF3, IFNß and IL-28b. The transbodies have high potential for testing further as a novel anti-HCV agent, either alone, adjunct of existing anti-HCV agents/remedies, or in combination with their cognates specific to other HCV enzymes/proteins.


Assuntos
Anticorpos Antivirais/administração & dosagem , Hepacivirus/imunologia , Hepacivirus/fisiologia , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/fisiologia , Replicação Viral/imunologia , Replicação Viral/fisiologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/genética , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Antivirais/administração & dosagem , Antivirais/química , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Técnicas de Visualização da Superfície Celular , Peptídeos Penetradores de Células/administração & dosagem , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Simulação por Computador , Hepacivirus/genética , Humanos , Imunidade Inata/genética , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Transfecção , Proteínas não Estruturais Virais/genética , Replicação Viral/genética
19.
Mol Pharm ; 13(12): 4034-4042, 2016 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-27749081

RESUMO

The effect of carrier morphology on the intestinal absorption of insulin was investigated using a morphology-tunable polymeric carrier, ß-cyclodextrin-grafted chitosan (BCC). The insulin-BCC complexes were prepared in either acetate or citrate buffer solutions, followed by dilution with phosphate buffer for the administration. The complex had a molecular network structure in the acetate buffer, whereas nanoparticles formed in the citrate buffer. The network structure in the acetate buffer was maintained even after dilution with a phosphate buffer, but the nanoparticles in the citrate buffer caused aggregation after dilution. Both complexes enhanced the intestinal absorption of insulin. Interestingly, their absorption profiles were totally different; prompt absorption was observed for the complex prepared in acetate buffer, whereas sustained absorption was observed for the complex prepared in citrate buffer. The difference in the absorption patterns was attributed to the difference in the complex morphology. Next, penetratin, a cell-penetrating peptide, was grafted to BCC to find further improvement in the absorption behavior. A simple mixture of penetratin and BCC was also effective. An oral administration study was also conducted in mice to observe effective suppression of glucose levels, which was further enhanced by coadministration of penetratin. Thus, BCC was proven to be an effective carrier for enhancing oral absorption of peptide drugs, and it is suggested that the carrier morphology is also an important factor that influences the absorption profile.


Assuntos
Proteínas de Transporte/administração & dosagem , Peptídeos Penetradores de Células/administração & dosagem , Quitosana/química , Portadores de Fármacos/química , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Absorção Intestinal/efeitos dos fármacos , beta-Ciclodextrinas/química , Administração Oral , Animais , Disponibilidade Biológica , Proteínas de Transporte/química , Peptídeos Penetradores de Células/química , Sistemas de Liberação de Medicamentos , Hipoglicemiantes/química , Insulina/química , Masculino , Camundongos , Nanopartículas/química , Ratos , Ratos Sprague-Dawley
20.
Subcell Biochem ; 76: 323-41, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26219718

RESUMO

Hypophosphatasia (HPP) is a rare monogenetic and multisystemic disease with involvement of different organs, including bone, muscle, kidney, lung, gastrointestinal tract and the nervous system. The exact metabolic mechanisms of the effects of TNAP deficiency in different tissues are not understood in detail. There is no approved specific treatment for HPP; therefore symptomatic treatment in order to improve the clinical features is of major interest. Enzyme replacement therapy (ERT) is a relatively new type of treatment based on the principle of administering a medical treatment replacing a defective or absent enzyme. Recently ERT with a bone targeted recombinant human TNAP molecule has been reported to be efficient in ten severely affected patients and improved survival of life threatening forms. These results are very promising especially with regard to the skeletal phenotype but it is unclear whether ERT also has beneficial effects for craniosynostosis and in other affected tissues in HPP such as brain and kidney. Long-term data are not yet available and further systematic clinical trials are needed. It is also necessary to establish therapeutic approaches to help patients who are affected by less severe forms of HPP but also suffer from a significant reduction in quality of life. Further basic research on TNAP function and role in different tissues and on its physiological substrates is critical to gain a better insight in the pathogenesis in HPP. This and further experiences in new therapeutic strategies may improve the prognosis and quality of life of patients with all forms of HPP.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/uso terapêutico , Terapia de Reposição de Enzimas , Hipofosfatasia/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Fosfatase Alcalina/administração & dosagem , Fosfatase Alcalina/uso terapêutico , Animais , Proteínas de Transporte/administração & dosagem , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Terapia de Reposição de Enzimas/métodos , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/uso terapêutico , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/uso terapêutico
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