RESUMEN
BACKGROUND: Sarcoid tumors are common in horses and may negatively impact the performance and value of the horse. No known treatment is reliably successful. HYPOTHESES/OBJECTIVES: To determine tolerability, overall response rate, time to response, and progression-free survival of horses with biopsy-confirmed or suspected sarcoids treated with ALVAC-fIL2. ANIMALS: Client-owned horses with measurable, presumed- or biopsy-confirmed sarcoid tumors. METHODS: Prospective pilot study. One milliliter of ALVAC-fIL2 was injected into 4 to 5 areas of the sarcoid(s) in each horse (week 0); this treatment was repeated in weeks 1, 3, and 7. Sarcoids were measured at each visit, and response to treatment was determined according to the Response Evaluation Criteria in Solid Tumors for dogs (v1.0). After the final treatment, horses were reassessed and sarcoids remeasured every 3 months until tumor progression or for a minimum of 1 year if progression was not documented. RESULTS: Fourteen horses were included. Tumor size decreased in 86% of the horses, and the median time to first response was 89 days (range, 34-406 days). Median time to best response was 211 days (range, 56-406 days), but 3 of the sarcoids still were decreasing in size at the time of final evaluation. The median progression-free interval was not reached. Adverse events were minimal and included transient focal inflammation in 2 horses. CONCLUSIONS AND CLINICAL IMPORTANCE: Intratumoral injection of ALVAC-fIL2 has promise as a well-tolerated and effective, tissue-sparing treatment for horses with sarcoid tumors.
Asunto(s)
Enfermedades de los Caballos , Factores Inmunológicos , Sarcoidosis , Adyuvantes Inmunológicos , Animales , Enfermedades de los Caballos/tratamiento farmacológico , Caballos , Factores Inmunológicos/uso terapéutico , Interleucina-2 , Proyectos Piloto , Estudios Prospectivos , Sarcoidosis/tratamiento farmacológico , Sarcoidosis/veterinariaRESUMEN
A small molecule called EMD 57033 can repair motor proteins that have stopped working as a result of stress.
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Miosinas Cardíacas/metabolismo , Cardiotónicos/farmacología , Activadores de Enzimas/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Quinolinas/farmacología , Tiadiazinas/farmacología , Animales , HumanosRESUMEN
Alterations in the lipid composition of endosomal-lysosomal membranes may constitute an early event in Alzheimer's disease (AD) pathogenesis. In this study, we investigated the possibility that GM2 ganglioside accumulation in a mouse model of Sandhoff disease might be associated with the accumulation of intraneuronal and extracellular proteins commonly observed in AD. Our results show intraneuronal accumulation of amyloid-ß peptide (Aß)-like, α-synuclein-like, and phospho-tau-like immunoreactivity in the brains of ß-hexosaminidase knock-out (HEXB KO) mice. Biochemical and immunohistochemical analyses confirmed that at least some of the intraneuronal Aß-like immunoreactivity (iAß-LIR) represents amyloid precursor protein C-terminal fragments (APP-CTFs) and/or Aß. In addition, we observed increased levels of Aß40 and Aß42 peptides in the lipid-associated fraction of HEXB KO mouse brains, and intraneuronal accumulation of ganglioside-bound Aß (GAß) immunoreactivity in a brain region-specific manner. Furthermore, α-synuclein and APP-CTFs and/or Aß were found to accumulate in different regions of the substantia nigra, indicating different mechanisms of accumulation or turnover pathways. Based on the localization of the accumulated iAß-LIR to endosomes, lysosomes, and autophagosomes, we conclude that a significant accumulation of iAß-LIR may be associated with the lysosomal-autophagic turnover of Aß and fragments of APP-containing Aß epitopes. Importantly, intraneuronal GAß immunoreactivity, a proposed prefibrillar aggregate found in AD, was found to accumulate throughout the frontal cortices of postmortem human GM1 gangliosidosis, Sandhoff disease, and Tay-Sachs disease brains. Together, these results establish an association between the accumulation of gangliosides, autophagic vacuoles, and the intraneuronal accumulation of proteins associated with AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Gangliósidos/metabolismo , Hexosaminidasa B/genética , Lisosomas/fisiología , Enfermedad de Sandhoff/patología , Adulto , Animales , Western Blotting , Química Encefálica/genética , Química Encefálica/fisiología , Preescolar , Gangliósido G(M2)/metabolismo , Humanos , Inmunohistoquímica , Lactante , Metabolismo de los Lípidos , Bulbo Raquídeo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Médula Espinal/metabolismo , Sustancia Negra/metabolismo , Adulto Joven , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMEN
Kaposi sarcoma (KS) is associated with human herpesvirus (HHV)-8 and is dependent on the induction of vascular endothelial growth factors (VEGFs). VEGF regulates genes that provide arterial or venous identity to endothelial cells, such as the induction of EphrinB2, which phenotypically defines arterial endothelial cells and pericytes, and represses EphB4, which defines venous endothelial cells. We conducted a comprehensive analysis of the Eph receptor tyrosine kinases to determine which members are expressed and therefore contribute to KS pathogenesis. We demonstrated limited Eph/Ephrin expression; notably, the only ligand highly expressed is EphrinB2. We next studied the biologic effects of blocking EphrinB2 using the extracellular domain of EphB4 fused with human serum albumin (sEphB4-HSA). sEphB4-HSA inhibited migration and invasion of the KS cells in vitro in response to various growth factors. Finally, we determined the biologic effects of combining sEphB4-HSA and an antibody to VEGF. sEphB4-HSA was more active than the VEGF antibody, and combination of the 2 had at least additive activity. sEphB4-HSA reduced blood vessel density, pericyte recruitment, vessel perfusion, and increased hypoxia, with an associated increase in VEGF and DLL4 expression. The combination of sEphB4-HSA and VEGF antibody is a rational treatment combination for further investigation.
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Efrina-B2/antagonistas & inhibidores , Efrina-B2/metabolismo , Receptor EphB4/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Sarcoma de Kaposi/fisiopatología , Neoplasias Vasculares/fisiopatología , Animales , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Células Endoteliales/citología , Efrinas/genética , Efrinas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Receptor EphB4/genética , Receptores de la Familia Eph/genética , Receptores de la Familia Eph/metabolismo , Proteínas Recombinantes de Fusión/genética , Sarcoma de Kaposi/tratamiento farmacológico , Sarcoma de Kaposi/metabolismo , Albúmina Sérica/genética , Arterias Umbilicales/citología , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular/inmunología , Neoplasias Vasculares/tratamiento farmacológico , Neoplasias Vasculares/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The highly glycosylated peptide hormone erythropoietin (EPO) plays a key role in the regulation of erythrocyte maturation. Currently, marketed EPO is produced by recombinant technology in mammalian cell cultures. The complementary DNA (cDNA) for human EPO (hEPO) was transiently and stably expressed in the moss Physcomitrella patens wild-type and Delta-fuc-t Delta-xyl-t mutant, the latter containing N-glycans lacking the plant-specific, core-bound alpha1,3-fucose and beta1,2-xylose. New expression vectors were designed based on a Physcomitrella ubiquitin gene-derived promoter for the expression of hEPO cDNA. Transient expression in protoplasts was much stronger at 10 than at 20 degrees C. In Western blot analysis, the molecular size of moss-produced recombinant human EPO (rhEPO) was identified to be 30 kDa, and it accumulated in the medium of transiently transformed protoplasts to high levels around 0.5 microg/mL. Transgenic Physcomitrella Delta-fuc-t Delta-xyl-t mutant lines expressing EPO cDNA showed secretion of rhEPO through the cell wall to the culture medium. In 5- and 10-L photobioreactor cultures, secreted rhEPO accumulated to high levels above 250 microg/g dry weight of moss material after 6 days. Silver staining of rhEPO on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) taken from the bioreactor culture demonstrated a high purity of the over-expressed secreted rhEPO, with a very low background of endogenous moss proteins. Peptide mapping of rhEPO produced by the Physcomitrella Delta-fuc-t Delta-xyl-t mutant indicated correct processing of the plant-derived signal peptide. All three N-glycosylation sites of rhEPO were occupied by complex-type N-glycans completely devoid of the plant-specific core sugar residues fucose and xylose.
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Bryopsida/genética , Eritropoyetina/metabolismo , Mutación , Reactores Biológicos , Western Blotting , Bryopsida/metabolismo , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Eritropoyetina/genética , Glicosilación , Humanos , Mapeo Peptídico , Plantas Modificadas Genéticamente/metabolismo , Protoplastos/metabolismo , Proteínas Recombinantes , Temperatura , Transformación GenéticaRESUMEN
Covalent attachment of polyethylene glycol, PEGylation, has been shown to prolong the half-life and enhance the pharmacodynamics of therapeutic proteins. Current methods for PEGylation, which rely on chemical conjugation through reactive groups on amino acids, often generate isoforms in which PEG is attached at sites that interfere with bioactivity. Here, we present a novel strategy for site-directed PEGylation using glycosyltransferases to attach PEG to O-glycans. The process involves enzymatic GalNAc glycosylation at specific serine and threonine residues in proteins expressed without glycosylation in Escherichia coli, followed by enzymatic transfer of sialic acid conjugated with PEG to the introduced GalNAc residues. The strategy was applied to three therapeutic polypeptides, granulocyte colony stimulating factor (G-CSF), interferon-alpha2b (IFN-alpha2b), and granulocyte/macrophage colony stimulating factor (GM-CSF), which are currently in clinical use.