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1.
Radiat Res ; 192(2): 159-168, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31188068

RESUMO

In this work, we investigated the change in tumor microenvironment caused by semi-ablative high-dose irradiation and its implication on tumor cell survival, reoxygenation of hypoxic cells and repopulation in FSaII tumors grown subcutaneously in the hind legs of C3H mice. Tumors were exposed to 10-30 Gy of X-ray radiation in a single exposure, and the vascularity and blood perfusion were assessed based on the levels of CD31 expression and Hoechst 33342 perfusion, respectively. The tumor hypoxia was assessed by staining for pimonidazole adduct formation and the expression of hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase 9 (CA9). Tumor cell survival was determined using in vivo-in vitro excision assay method. The proportion of hypoxic cells in the tumor was determined from the surviving cell fraction in tumors exposed to a test dose under aerobic and hypoxic conditions. Radiation expsoure markedly reduced the functional vascularity and blood perfusion, and profoundly increased the expression of HIF-1α and CA9 pointing to an increase in tumor hypoxia. The overall clonogenic cell survival progressively decreased during 2-5 days postirradiation, most likely due to the radiation-induced vascular dysfunction. In turn, the proportion of surviving hypoxic cells decreased over several days postirradiation, presumably due to reoxygenation of hypoxic cells. The oxygen supplied through small fractions of blood vessels that survived the high-dose exposure, together with a reduction of oxygen consumption due to massive cell death, appeared to be the cause of the reoxygenation of hypoxic cells. The surviving tumor cells then subsequently repopulated. The findings from this study using a murine tumor model suggest that the efficacy of stereotactic body radiotherapy (SBRT) and stereotactic radiosurgery (SRS) may be significantly improved by allowing an inter-fraction time for reoxygenation while avoiding repopulation.

2.
Cell Host Microbe ; 25(1): 39-48.e5, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30629917

RESUMO

Passive administration of monoclonal antibodies (mAbs) is a promising therapeutic approach for Ebola virus disease (EVD). However, all mAbs and mAb cocktails that have entered clinical development are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against outbreak-causing Bundibugyo virus (BDBV) and Sudan virus (SUDV). Here, we advance MBP134, a cocktail of two broadly neutralizing human mAbs, ADI-15878 from an EVD survivor and ADI-23774 from the same survivor but specificity-matured for SUDV GP binding affinity, as a candidate pan-ebolavirus therapeutic. MBP134 potently neutralized all ebolaviruses and demonstrated greater protective efficacy than ADI-15878 alone in EBOV-challenged guinea pigs. A second-generation cocktail, MBP134AF, engineered to effectively harness natural killer (NK) cells afforded additional improvement relative to its precursor in protective efficacy against EBOV and SUDV in guinea pigs. MBP134AF is an optimized mAb cocktail suitable for evaluation as a pan-ebolavirus therapeutic in nonhuman primates.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Bem-Estar do Animal , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/uso terapêutico , Antivirais , Modelos Animais de Doenças , Ebolavirus/patogenicidade , Epitopos/imunologia , Feminino , Filoviridae/imunologia , Cobaias , Doença pelo Vírus Ebola/virologia , Humanos , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Recombinantes/imunologia , Resultado do Tratamento
3.
Sci Transl Med ; 9(384)2017 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-28381540

RESUMO

As observed during the 2013-2016 Ebola virus disease epidemic, containment of filovirus outbreaks is challenging and made more difficult by the lack of approved vaccine or therapeutic options. Marburg and Ravn viruses are highly virulent and cause severe and frequently lethal disease in humans. Monoclonal antibodies (mAbs) are a platform technology in wide use for autoimmune and oncology indications. Previously, we described human mAbs that can protect mice from lethal challenge with Marburg virus. We demonstrate that one of these mAbs, MR191-N, can confer a survival benefit of up to 100% to Marburg or Ravn virus-infected rhesus macaques when treatment is initiated up to 5 days post-inoculation. These findings extend the small but growing body of evidence that mAbs can impart therapeutic benefit during advanced stages of disease with highly virulent viruses and could be useful in epidemic settings.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Infecções por Filoviridae/tratamento farmacológico , Filoviridae/fisiologia , Doença do Vírus de Marburg/tratamento farmacológico , Marburgvirus/fisiologia , Animais , Proteção Cruzada , Infecções por Filoviridae/virologia , Cobaias , Humanos , Macaca fascicularis , Macaca mulatta , Doença do Vírus de Marburg/virologia , Projetos Piloto
4.
Virology ; 490: 49-58, 2016 03.
Artigo em Inglês | MEDLINE | ID: mdl-26828465

RESUMO

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was identified in 2012 as the causative agent of a severe, lethal respiratory disease occurring across several countries in the Middle East. To date there have been over 1600 laboratory confirmed cases of MERS-CoV in 26 countries with a case fatality rate of 36%. Given the endemic region, it is possible that MERS-CoV could spread during the annual Hajj pilgrimage, necessitating countermeasure development. In this report, we describe the clinical and radiographic changes of rhesus monkeys following infection with 5×10(6) PFU MERS-CoV Jordan-n3/2012. Two groups of NHPs were treated with either a human anti-MERS monoclonal antibody 3B11-N or E410-N, an anti-HIV antibody. MERS-CoV Jordan-n3/2012 infection resulted in quantifiable changes by computed tomography, but limited other clinical signs of disease. 3B11-N treated subjects developed significantly reduced lung pathology when compared to infected, untreated subjects, indicating that this antibody may be a suitable MERS-CoV treatment.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/patologia , Pulmão/patologia , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Animais , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Humanos , Pulmão/virologia , Macaca mulatta , Masculino
6.
PLoS Pathog ; 11(6): e1005016, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26115029

RESUMO

The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel "wing" feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Doença pelo Vírus Ebola/imunologia , Imunização , Doença do Vírus de Marburg/prevenção & controle , Marburgvirus/imunologia , Animais , Anticorpos Antivirais/imunologia , Reações Cruzadas/imunologia , Ebolavirus/imunologia , Feminino , Masculino , Doença do Vírus de Marburg/imunologia , Camundongos Endogâmicos BALB C
7.
J Immunol Methods ; 422: 111-7, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25865265

RESUMO

We have produced and characterized two chimeric human IgG1 monoclonal antibodies that bind different immunodominant epitopes on Vibrio cholerae lipopolysaccharide (LPS). MAb 2D6 IgG1 recognizes Ogawa O-polysaccharide antigen, while mAb ZAC-3 IgG1 recognizes core/lipid A moiety of Ogawa and Inaba LPS. Both antibodies were expressed using a Nicotiana benthamiana-based rapid antibody-manufacturing platform (RAMP) and evaluated in vitro for activities associated with immunity to V. cholerae, including vibriocidal activity, bacterial agglutination and motility arrest.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Epitopos Imunodominantes/imunologia , Imunoglobulina G/imunologia , Vibrio cholerae/imunologia , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/genética , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Antígenos de Bactérias/imunologia , Quimera/imunologia , Clonagem Molecular , Epitopos/imunologia , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Lipopolissacarídeos/imunologia , Antígenos O/imunologia , Tabaco/genética
8.
Clin Vaccine Immunol ; 21(5): 777-82, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24574537

RESUMO

Recent incidents in the United States and abroad have heightened concerns about the use of ricin toxin as a bioterrorism agent. In this study, we produced, using a robust plant-based platform, four chimeric toxin-neutralizing monoclonal antibodies that were then evaluated for the ability to passively protect mice from a lethal-dose ricin challenge. The most effective antibody, c-PB10, was further evaluated in mice as a therapeutic following ricin exposure by injection and inhalation.


Assuntos
Antitoxinas/administração & dosagem , Imunização Passiva/métodos , Planticorpos/administração & dosagem , Envenenamento/prevenção & controle , Ricina/antagonistas & inibidores , Ricina/toxicidade , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Feminino , Camundongos Endogâmicos BALB C , Análise de Sobrevida , Resultado do Tratamento
9.
Sci Transl Med ; 5(199): 199ra113, 2013 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-23966302

RESUMO

Ebola virus (EBOV) remains one of the most lethal transmissible infections and is responsible for high fatality rates and substantial morbidity during sporadic outbreaks. With increasing human incursions into endemic regions and the reported possibility of airborne transmission, EBOV is a high-priority public health threat for which no preventive or therapeutic options are currently available. Recent studies have demonstrated that cocktails of monoclonal antibodies are effective at preventing morbidity and mortality in nonhuman primates (NHPs) when administered as a post-exposure prophylactic within 1 or 2 days of challenge. To test whether one of these cocktails (MB-003) demonstrates efficacy as a therapeutic (after the onset of symptoms), we challenged NHPs with EBOV and initiated treatment upon confirmation of infection according to a diagnostic protocol for U.S. Food and Drug Administration Emergency Use Authorization and observation of a documented fever. Of the treated animals, 43% survived challenge, whereas both the controls and all historical controls with the same challenge stock succumbed to infection. These results represent successful therapy of EBOV infection in NHPs.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Vacinas contra Ebola/uso terapêutico , Ebolavirus/imunologia , Doença pelo Vírus Ebola/terapia , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Modelos Animais de Doenças , Vacinas contra Ebola/administração & dosagem , Ebolavirus/genética , Feminino , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Macaca mulatta , Masculino , Planticorpos/administração & dosagem , Planticorpos/uso terapêutico , Profilaxia Pós-Exposição/métodos , Pesquisa Médica Translacional , Viremia/imunologia , Viremia/prevenção & controle , Viremia/terapia
10.
MAbs ; 5(2): 263-9, 2013 Mar-Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23396091

RESUMO

Severe lower respiratory tract infection in infants and small children is commonly caused by respiratory syncytial virus (RSV). Palivizumab (Synagis(®)), a humanized IgG1 monoclonal antibody (mAb) approved for RSV immunoprophylaxis in at-risk neonates, is highly effective, but pharmacoeconomic analyses suggest its use may not be cost-effective. Previously described potent RSV neutralizers (human Fab R19 and F2-5; human IgG RF-1 and RF-2) were produced in IgG format in a rapid and inexpensive Nicotiana-based manufacturing system for comparison with palivizumab. Both plant-derived (palivizumab-N) and commercial palivizumab, which is produced in a mouse myeloma cell line, showed protection in prophylactic (p < 0.001 for both mAbs) and therapeutic protocols (p < 0.001 and p < 0.05 respectively). The additional plant-derived human mAbs directed against alternative epitopes displayed neutralizing activity, but conferred less protection in vivo than palivizumab-N or palivizumab. Palivizumab remains one of the most efficacious RSV mAbs described to date. Production in plants may reduce manufacturing costs and improve the pharmacoeconomics of RSV immunoprophylaxis and therapy.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Vírus Sincicial Respiratório Humano/imunologia , Tabaco/imunologia , Animais , Anticorpos Monoclonais Humanizados/economia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/economia , Anticorpos Neutralizantes/imunologia , Modelos Animais de Doenças , Humanos , Palivizumab , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/virologia , Sigmodontinae , Resultado do Tratamento
11.
Toxicon ; 60(1): 83-94, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22497788

RESUMO

Milked venom from cone snails represent a novel biological resource with a proven track record for drug discovery. To strengthen this correlation, we undertook a chromatographic and mass spectrometric study of individual milked venoms from Conus purpurascens. Milked venoms demonstrate extensive peptide differentiation amongst individual specimens and during captivity. Individual snails were found to lack a consistent set of described conopeptides, but instead demonstrated the ability to change venom expression, composition and post-translational modification incorporation; all variations contribute to an increase in chemical diversity and prey targeting strategies. Quantitative amino acid analysis revealed that milked venom peptides are expressed at ranges up to 3.51-121.01 µM within single milked venom samples. This provides for a 6.37-20,965 fold-excess of toxin to induce apparent IC50 for individual conopeptides identified in this study. Comparative molecular mass analysis of duct venom, milked venom and radula tooth extracts from single C. purpurascens specimens demonstrated a level of peptide continuity. Numerous highly abundant and unique conopeptides remain to be characterized. This study strengthens the notion that approaches in conopeptide drug lead discovery programs will potentially benefit from a greater understanding of the toxinological nature of the milked venoms of Conus.


Assuntos
Caramujo Conus , Venenos de Moluscos , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Peso Molecular , Venenos de Moluscos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta
12.
BMC Cell Biol ; 12: 7, 2011 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-21272378

RESUMO

BACKGROUND: Annexins are calcium dependent phospholipid binding proteins that are expressed in a wide variety of tissues and implicated in various extra- and intracellular processes. In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal. Conflicting reports from transgenic mice overexpressing annexin A6 or null mice lacking annexin A6 showed imbalances in intracellular calcium turnover and disturbed cardiac contractility. However, few studies have focussed on the signalling module of annexin A6 in the heart either in normal or in pathological state. RESULTS: To identify the putative binding partners of annexin A6 in the heart, ventricular extracts were subjected to glutathione S-transferase (GST)- annexin A6 pull down assay and the GST- annexin A6 bound proteins were identified by mass spectrometry. The pull down fractions of ventricular extracts with GST-full length annexin A6 as well as GST-C terminus deleted annexin A6 when immunoblotted with anti sarcomeric alpha (α)-actinin antibody showed the presence of α-actinin in the immunoblot which was absent when GST-N terminus deleted annexin A6 was used for pull down. Overexpression of green fluorescent protein (GFP) tagged full length annexin A6 showed z-line like appearance in cardiomyocytes whereas GFP-N termimus deleted annexin A6 was mostly localized to the nucleus. Overexpression of GFP-C terminus deleted annexin A6 in cardiomyocytes showed aggregate like appearance in the cytoplasm. Double immunofluorescent staining of cardiomyocytes with anti annexin A6 and anti sarcomeric α-actinin antibodies showed perfect co-localization of these two proteins with annexin A6 appearing like a component of sarcomere. Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells. CONCLUSIONS: In overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.


Assuntos
Actinina/metabolismo , Anexina A6/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Anexina A6/análise , Anexina A6/genética , Anticorpos/imunologia , Células Cultivadas , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/citologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Sarcômeros/metabolismo , Transdução de Sinais
13.
Biochem Biophys Res Commun ; 289(4): 882-7, 2001 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-11735129

RESUMO

Junctate is a newly identified integral ER/SR membrane calcium binding protein, which is an alternative splicing form of the same gene generating aspartyl beta-hydroxylase and junctin. Screening a mouse heart cDNA library using canine junctin cDNA as a probe yielded three complete mouse heart cDNAs. One of the cDNAs is homologous to the previously reported human junctate. The three mouse junctate proteins are composed of 270, 259, and 215 amino acids (we named them junctate-1, -2, and -3). The apparent molecular masses of the mouse junctates in SDS-PAGE were in the range between 40 and 53 kDa. Northern and Western blot analyses indicate that mouse junctates are expressed in heart, brain, spleen, lung, liver, kidney, and stomach, but not in skeletal muscle. The apparent molecular weights of junctates from heart and brain were somewhat different from those from the other tissues tested, suggesting that there are tissue-specific expression patterns of the different junctate isoforms. Immunohistochemical studies showed that junctates were expressed both in ventricular and atrial tissues. This is the first study that shows the presence of 3 distinct cardiac junctate isoforms expressed in various mammalian tissues.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Oxigenases de Função Mista , Proteínas Musculares , Miocárdio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , DNA Complementar/genética , Cães , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Peso Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Homologia de Sequência de Aminoácidos , Distribuição Tecidual
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