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BACKGROUND: Interhospital transfer for patients with stroke due to large vessel occlusion for endovascular thrombectomy (EVT) has been associated with treatment delays. METHODS: We analyzed data from Optimizing Patient Treatment in Major Ischemic Stroke With EVT, a quality improvement registry to support EVT implementation in Canada. We assessed for unadjusted differences in baseline characteristics, time metrics, and procedural outcomes between patients with large vessel occlusion transferred for EVT and those directly admitted to an EVT-capable center. RESULTS: Between January 1, 2018, and December 31, 2021, a total of 6803 patients received EVT at 20 participating centers (median age, 73 years; 50% women; and 50% treated with intravenous thrombolysis). Patients transferred for EVT (n=3376) had lower rates of M2 occlusion (22% versus 27%) and higher rates of basilar occlusion (9% versus 5%) compared with those patients presenting directly at an EVT-capable center (n=3373). Door-to-needle times were shorter in patients receiving intravenous thrombolysis before transfer compared with those presenting directly to an EVT center (32 versus 36 minutes). Patients transferred for EVT had shorter door-to-arterial access times (37 versus 87 minutes) but longer last seen normal-to-arterial access times (322 versus 181 minutes) compared with those presenting directly to an EVT-capable center. No differences in arterial access-to-reperfusion times, successful reperfusion rates (85% versus 86%), or adverse periprocedural events were found between the 2 groups. Patients transferred to EVT centers had a similar likelihood for good functional outcome (modified Rankin Scale score, 0-2; 41% versus 43%; risk ratio, 0.95 [95% CI, 0.88-1.01]; adjusted risk ratio, 0.98 [95% CI, 0.91-1.05]) and a higher risk for all-cause mortality at 90 days (29% versus 25%; risk ratio, 1.15 [95% CI, 1.05-1.27]; adjusted risk ratio, 1.14 [95% CI, 1.03-1.28]) compared with patients presenting directly to an EVT center. CONCLUSIONS: Patients transferred for EVT experience significant delays from the time they were last seen normal to the initiation of EVT.
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Procedimientos Endovasculares , Accidente Cerebrovascular Isquémico , Transferencia de Pacientes , Sistema de Registros , Trombectomía , Tiempo de Tratamiento , Humanos , Femenino , Masculino , Anciano , Procedimientos Endovasculares/métodos , Canadá/epidemiología , Persona de Mediana Edad , Anciano de 80 o más Años , Trombectomía/métodos , Accidente Cerebrovascular Isquémico/cirugía , Accidente Cerebrovascular Isquémico/terapia , Accidente Cerebrovascular/terapia , Accidente Cerebrovascular/cirugía , Accidente Cerebrovascular/epidemiología , Terapia Trombolítica/métodos , Resultado del TratamientoRESUMEN
Structure and function of therapeutic antibodies can be modulated by a variety of post-translational modifications (PTM). Tyrosine (Tyr) sulfation is a type of negatively charged PTM that occurs during protein trafficking through the Golgi. In this study, we discovered that an anti-interleukin (IL)-4 human IgG1, produced by transiently transfected HEK293 cells, contained a fraction of unusual negatively charged species. Interestingly, the isolated acidic species exhibited a two-fold higher affinity to IL-4 and a nearly four-fold higher potency compared to the main species. Mass spectrometry (MS) showed the isolated acidic species possessed an +80-Dalton from the expected mass, suggesting an occurrence of Tyr sulfation. Consistent with this hypothesis, we show the ability to control the acidic species during transient expression with the addition of Tyr sulfation inhibitor sodium chlorate or, conversely, enriched the acidic species from 30% to 92% of the total antibody protein when the IL-4 IgG was co-transfected with tyrosylprotein sulfotransferase genes. Further MS and mutagenesis analysis identified a Tyr residue at the light chain complementarity-determining region-1 (CDRL-1), which was sulfated specifically. These results together have demonstrated for the first time that Tyr sulfation at CDRL-1 could modulate antibody binding affinity and potency to a human immune cytokine.
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Interleucina-4 , Tirosina , Humanos , Tirosina/metabolismo , Células HEK293 , Aparato de Golgi/metabolismo , MutagénesisRESUMEN
The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and ß-glucuronidase assays in vitro and distinct properties in vivo Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N'-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease.
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Glucuronidasa/metabolismo , Insuficiencia Renal Crónica/patología , Animales , Células CHO , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , Factor-23 de Crecimiento de Fibroblastos , Tasa de Filtración Glomerular/efectos de los fármacos , Glucuronidasa/química , Glucuronidasa/genética , Glicopéptidos/análisis , Células HEK293 , Semivida , Humanos , Proteínas Klotho , Espectrometría de Masas , Mutagénesis Sitio-Dirigida , Procesamiento Proteico-Postraduccional , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Insuficiencia Renal Crónica/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/patología , Daño por Reperfusión/veterinaria , Relación Estructura-ActividadRESUMEN
BACKGROUND: The optimal management of tandem carotid lesions during endovascular thrombectomy (EVT) remains uncertain. The safety and efficacy of acute carotid artery stenting (aCAS) are debated, including safety concerns such as procedural complications and symptomatic intracerebral hemorrhage (sICH). We aimed to assess aCAS safety among EVT-treated patients using a large Canadian registry. METHODS: We retrospectively analyzed the OPTIMISE registry and compared adult patients undergoing EVT and aCAS versus EVT only. The primary outcome was a composite of in-hospital death, long-term care facility destination at discharge, sICH, or any EVT-related procedural complications. Secondary outcomes included individual components of the primary outcome, EVT workflow times, final modified Thrombolysis in Cerebral Ischemia score and 90-day modified Rankin Scale score. Statistical significance was evaluated by a multivariate logistic regression model. RESULTS: 4205 patients were included (330 with EVT-aCAS and 3875 with EVT-only). Both groups were similar with regard to baseline National Institutes of Health Stroke Scale score, Alberta Stroke Program Early CT Score and use of IV thrombolysis, but differed in age (EVT-aCAS group 67.2±12.1 years vs EVT-only group 71.3±14.1 years, P<0.001), proportion of women (28.2% vs 53.3%, P<0.001), and occlusion location (internal carotid artery terminus 44% vs 16%, P<0.001). The EVT-aCAS group showed a non-significant increase in odds of composite safety outcomes (adjusted OR 1.35 (95% CI 0.97 to 1.84), P=0.06) with a significantly higher proportion of procedural complications (10.0% vs 6.2%, P=0.002). CONCLUSION: In a large national registry, EVT-aCAS was associated with a higher proportion of unfavorable safety outcomes, driven by more frequent procedural complications. Further research is needed to clarify the role of aCAS in tandem occlusion stroke.
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The Ascyrus Medical Dissection Stent (AMDS) has proved to promote false lumen closure at the distal anastomosis in type A dissection repairs. During the past 20 years, open techniques have developed into endovascular repairs. We report a case of thoracic endovascular aortic repair (TEVAR) landed inside an AMDS. A 63-year-old man with hypertension presented with type A aortic dissection. After initial repair with AMDS, he returned for TEVAR to fix residual type B dissection. A proper seal was created between the AMDS and TEVAR stents, demonstrating a successful off-label use of the AMDS stent.
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Aneurisma de la Aorta Torácica , Implantación de Prótesis Vascular , Procedimientos Endovasculares , Masculino , Humanos , Persona de Mediana Edad , Aneurisma de la Aorta Torácica/diagnóstico , Aneurisma de la Aorta Torácica/cirugía , Prótesis Vascular , Diseño de Prótesis , Resultado del Tratamiento , Factores de Tiempo , Stents , Estudios RetrospectivosRESUMEN
For an antibody to be a successful therapeutic many competing factors require optimization, including binding affinity, biophysical characteristics, and immunogenicity risk. Additional constraints may arise from the need to formulate antibodies at high concentrations (>150 mg/ml) to enable subcutaneous dosing with reasonable volume (ideally <1.0 mL). Unfortunately, antibodies at high concentrations may exhibit high viscosities that place impractical constraints (such as multiple injections or large needle diameters) on delivery and impede efficient manufacturing. Here we describe the optimization of an anti-PDGF-BB antibody to reduce viscosity, enabling an increase in the formulated concentration from 80 mg/ml to greater than 160 mg/ml, while maintaining the binding affinity. We performed two rounds of structure guided rational design to optimize the surface electrostatic properties. Analysis of this set demonstrated that a net-positive charge change, and disruption of negative charge patches were associated with decreased viscosity, but the effect was greatly dependent on the local surface environment. Our work here provides a comprehensive study exploring a wide sampling of charge-changes in the Fv and CDR regions along with targeting multiple negative charge patches. In total, we generated viscosity measurements for 40 unique antibody variants with full sequence information which provides a significantly larger and more complete dataset than has previously been reported.
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Anticuerpos Monoclonales/química , Inmunoglobulina G/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Becaplermina/inmunología , Diseño Asistido por Computadora , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Modelos Moleculares , Mutación , Conformación Proteica , Propiedades de Superficie , ViscosidadRESUMEN
BACKGROUND: Intercostal artery aneurysms are rare vascular abnormalities that are typically diagnosed following rupture in patients with predisposing conditions. Our report is the first to document a patient with unruptured intercostal artery aneurysms in the absence of any associated disease. CASE PRESENTATION: A 70-year-old male with prostatic adenocarcinoma was incidentally discovered to have multiple unruptured aneurysms of his intercostal arteries. Three of the aneurysms were embolized utilizing microcoils and glue. At six-month follow-up the patient remained asymptomatic. CONCLUSION: We demonstrate successful endovascular management of a unique case of multiple idiopathic unruptured intercostal artery aneurysms. Appropriate diagnosis and prompt treatment of these rare vascular lesions is essential in preventing the potentially catastrophic consequences of rupture.
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Advancement in high-throughput screening methods of novel therapeutic proteins for early stage research and development, specifically mAbs, have given mid-scale (milligram to gram scale) purification groups access to more of these molecules. The available purification technologies built to support mid-scale production was not efficient or versatile enough to keep up with this surge. To remedy this problem, we have designed and built a custom instrument using an ÄKTA Pure. This system enables parallel processing up to 5 samples and has the versatility to perform 1- to 3-step purification processes in a single queue. Furthermore, a unique purification scheme can be selected for each of the five samples in the queue. Overall processing time has reduced by 83% compared to manual, non-parallel load methods. Here, we describe our novel approach and demonstrate the flexibility, speed and efficiency of the instrument.
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Anticuerpos Monoclonales/aislamiento & purificación , Química Farmacéutica/instrumentación , Química Farmacéutica/métodos , Cromatografía de AfinidadRESUMEN
Protein modifications by intricate cellular machineries often redesign the structure and function of existing proteins to impact biological networks. Disulfide bond formation between cysteine (Cys) pairs is one of the most common modifications found in extracellularly-destined proteins, key to maintaining protein structure. Unpaired surface cysteines on secreted mammalian proteins are also frequently found disulfide-bonded with free Cys or glutathione (GSH) in circulation or culture, the mechanism for which remains unknown. Here we report that these so-called Cys-capping modifications take place outside mammalian cells, not in the endoplasmic reticulum (ER) where oxidoreductase-mediated protein disulfide formation occurs. Unpaired surface cysteines of extracellularly-arrived proteins such as antibodies are uncapped upon secretion before undergoing disulfide exchange with cystine or oxidized GSH in culture medium. This observation has led to a feasible way to selectively modify the nucleophilic thiol side-chain of cell-surface or extracellular proteins in live mammalian cells, by applying electrophiles with a chemical handle directly into culture medium. These findings provide potentially an effective approach for improving therapeutic conjugates and probing biological systems.