RESUMEN
CD33 is a transmembrane receptor expressed on cells of myeloid lineage and regulates innate immunity. CD33 is a risk factor for Alzheimer's disease (AD) and targeting CD33 has been a promising strategy drug development. However, the mechanism of CD33's action is poorly understood. Here we investigate the mechanism of anti-CD33 antibody HuM195 (Lintuzumab) and its single-chain variable fragment (scFv) and examine their therapeutic potential. Treatment with HuM195 full-length antibody or its scFv increased phagocytosis of ß-amyloid 42 (Aß42) in human microglia and monocytes. This activation of phagocytosis was driven by internalization and degradation of CD33, thereby downregulating its inhibitory signal. HumM195 transiently induced CD33 phosphorylation and its signaling via receptor dimerization. However, this signaling decayed with degradation of CD33. scFv binding to CD33 leads to a degradation of CD33 without detection of the CD33 dimerization and signaling. Moreover, we found that treatments with either HuM195 or scFv promotes the secretion of IL33, a cytokine implicated in microglia reprogramming. Importantly, recombinant IL33 potentiates the uptake of Aß42 in monocytes. Collectively, our findings provide unanticipated mechanistic insight into the role of CD33 signaling in both monocytes and microglia and define a molecular basis for the development of CD33-based therapy of AD.
RESUMEN
The mechanism by which γ-secretase activating protein (GSAP) regulates γ-secretase activity has not yet been elucidated. Here, we show that knockout of GSAP in cultured cells directly reduces γ-secretase activity for Aß production, but not for Notch1 cleavage, suggesting that GSAP may induce a conformational change contributing to the specificity of γ-secretase. Furthermore, using an active-site-directed photoprobe with double cross-linking moieties, we demonstrate that GSAP modifies the orientation and/or distance of the PS1 N-terminal fragment and the PS1 C-terminal fragment, a region containing the active site of γ-secretase. This work offers insight into how GSAP regulates γ-secretase specificity.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Presenilina-1/química , Proteínas/metabolismo , Sistemas CRISPR-Cas , Dominio Catalítico , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Cinética , Fragmentos de Péptidos/metabolismo , Proteínas/genética , Receptor Notch1RESUMEN
BACKGROUND: Traumatic brain injury (TBI) is a major cause of death and disability. TBI results in a prolonged secondary central neuro-inflammatory response. Previously, we have demonstrated that multiple doses (2 and 24 h after TBI) of multipotent adult progenitor cells (MAPC) delivered intravenously preserve the blood-brain barrier (BBB), improve spatial learning, and decrease activated microglia/macrophages in the dentate gyrus of the hippocampus. In order to determine if there is an optimum treatment window to preserve the BBB, improve cognitive behavior, and attenuate the activated microglia/macrophages, we administered MAPC at various clinically relevant intervals. METHODS: We administered two injections intravenously of MAPC treatment at hours 2 and 24 (2/24), 6 and 24 (6/24), 12 and 36 (12/36), or 36 and 72 (36/72) post cortical contusion injury (CCI) at a concentration of 10 million/kg. For BBB experiments, animals that received MAPC at 2/24, 6/24, and 12/36 were euthanized 72 h post injury. The 36/72 treated group was harvested at 96 h post injury. RESULTS: Administration of MAPC resulted in a significant decrease in BBB permeability when administered at 2/24 h after TBI only. For behavior experiments, animals were harvested post behavior paradigm. There was a significant improvement in spatial learning (120 days post injury) when compared to cortical contusion injury (CCI) in groups when MAPC was administered at or before 24 h. In addition, there was a significant decrease in activated microglia/macrophages in the dentate gyrus of hippocampus of the treated group (2/24) only when compared to CCI. CONCLUSIONS: Intravenous injections of MAPC at or before 24 h after CCI resulted in improvement of the BBB, improved cognitive behavior, and attenuated activated microglia/macrophages in the dentate gyrus.
Asunto(s)
Lesiones Traumáticas del Encéfalo/cirugía , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Madre Multipotentes/fisiología , Animales , Barrera Hematoencefálica/fisiopatología , Proteínas de Unión al Calcio/metabolismo , Permeabilidad Capilar/fisiología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Proteínas de Dominio Doblecortina , Inyecciones Intraventriculares , Masculino , Aprendizaje por Laberinto , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Células Madre Multipotentes/trasplante , Neuropéptidos/metabolismo , Ratas , Tiempo de Reacción , Factores de TiempoRESUMEN
Preclinical studies using bone marrow derived cells to treat traumatic brain injury have demonstrated efficacy in terms of blood-brain barrier preservation, neurogenesis, and functional outcomes. Phase 1 clinical trials using bone marrow mononuclear cells infused intravenously in children with severe traumatic brain injury demonstrated safety and potentially a central nervous system structural preservation treatment effect. This study sought to confirm the safety, logistic feasibility, and potential treatment effect size of structural preservation/inflammatory biomarker mitigation in adults to guide Phase 2 clinical trial design. Adults with severe traumatic brain injury (Glasgow Coma Scale 5-8) and without signs of irreversible brain injury were evaluated for entry into the trial. A dose escalation format was performed in 25 patients: 5 controls, followed 5 patients in each dosing cohort (6, 9, 12 ×106 cells/kg body weight), then 5 more controls. Bone marrow harvest, cell processing to isolate the mononuclear fraction, and re-infusion occurred within 48 hours after injury. Patients were monitored for harvest-related hemodynamic changes, infusional toxicity, and adverse events. Outcome measures included magnetic resonance imaging-based measurements of supratentorial and corpus callosal volumes as well as diffusion tensor imaging-based measurements of fractional anisotropy and mean diffusivity of the corpus callosum and the corticospinal tract at the level of the brainstem at 1 month and 6 months postinjury. Functional and neurocognitive outcomes were measured and correlated with imaging data. Inflammatory cytokine arrays were measured in the plasma pretreatment, posttreatment, and at 1 and 6 month follow-up. There were no serious adverse events. There was a mild pulmonary toxicity of the highest dose that was not clinically significant. Despite the treatment group having greater injury severity, there was structural preservation of critical regions of interest that correlated with functional outcomes. Key inflammatory cytokines were downregulated. Treatment of severe, adult traumatic brain injury using an intravenously delivered autologous bone marrow mononuclear cell infusion is safe and logistically feasible. There appears to be a treatment signal as evidenced by central nervous system structural preservation, consistent with previous pediatric trial data. Inflammatory biomarkers are downregulated after cell infusion. Stem Cells 2016 Video Highlight: https://youtu.be/UiCCPIe-IaQ Stem Cells 2017;35:1065-1079.
Asunto(s)
Células de la Médula Ósea/citología , Lesiones Traumáticas del Encéfalo/terapia , Leucocitos Mononucleares/trasplante , Adulto , Conducta , Biomarcadores/sangre , Lesiones Traumáticas del Encéfalo/sangre , Lesiones Traumáticas del Encéfalo/patología , Cuerpo Calloso/patología , Citocinas/sangre , Femenino , Sustancia Gris/patología , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Tractos Piramidales/patología , Resultado del TratamientoRESUMEN
Mesenchymal stromal cells (MSCs) are believed to mobilize from the bone marrow in response to inflammation and injury, yet the effects of egress into the vasculature on MSC function are largely unknown. Here we show that wall shear stress (WSS) typical of fluid frictional forces present on the vascular lumen stimulates antioxidant and anti-inflammatory mediators, as well as chemokines capable of immune cell recruitment. WSS specifically promotes signaling through NFκB-COX2-prostaglandin E2 (PGE2 ) to suppress tumor necrosis factor-α (TNF-α) production by activated immune cells. Ex vivo conditioning of MSCs by WSS improved therapeutic efficacy in a rat model of traumatic brain injury, as evidenced by decreased apoptotic and M1-type activated microglia in the hippocampus. These results demonstrate that force provides critical cues to MSCs residing at the vascular interface which influence immunomodulatory and paracrine activity, and suggest the potential therapeutic use of force for MSC functional enhancement. Stem Cells 2017;35:1259-1272.
Asunto(s)
Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Administración Intravenosa , Animales , Antiinflamatorios/metabolismo , Fenómenos Biomecánicos , Reactores Biológicos , Lesiones Traumáticas del Encéfalo/patología , Lesiones Traumáticas del Encéfalo/terapia , Ciclooxigenasa 2/metabolismo , Dinoprostona/biosíntesis , Humanos , Inmunomodulación , Inflamación/patología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Fenotipo , Ratas , Reología , Transducción de Señal , Estrés MecánicoRESUMEN
OBJECTIVES: The devastating effect of traumatic brain injury is exacerbated by an acute secondary neuroinflammatory response, clinically manifest as elevated intracranial pressure due to cerebral edema. The treatment effect of cell-based therapies in the acute post-traumatic brain injury period has not been clinically studied although preclinical data demonstrate that bone marrow-derived mononuclear cell infusion down-regulates the inflammatory response. Our study evaluates whether pediatric traumatic brain injury patients receiving IV autologous bone marrow-derived mononuclear cells within 48 hours of injury experienced a reduction in therapeutic intensity directed toward managing elevated intracranial pressure relative to matched controls. DESIGN: The study was a retrospective cohort design comparing pediatric patients in a phase I clinical trial treated with IV autologous bone marrow-derived mononuclear cells (n = 10) to a control group of age- and severity-matched children (n = 19). SETTING: The study setting was at Children's Memorial Hermann Hospital, an American College of Surgeons Level 1 Pediatric Trauma Center and teaching hospital for the University of Texas Health Science Center at Houston from 2000 to 2008. PATIENTS: Study patients were 5-14 years with postresuscitation Glasgow Coma Scale scores of 5-8. INTERVENTIONS: The treatment group received 6 million autologous bone marrow-derived mononuclear cells/kg body weight IV within 48 hours of injury. The control group was treated in an identical fashion, per standard of care, guided by our traumatic brain injury management protocol, derived from American Association of Neurological Surgeons guidelines. MEASUREMENTS AND MAIN RESULTS: The primary measure was the Pediatric Intensity Level of Therapy scale used to quantify treatment of elevated intracranial pressure. Secondary measures included the Pediatric Logistic Organ Dysfunction score and days of intracranial pressure monitoring as a surrogate for length of neurointensive care. A repeated-measure mixed model with marginal linear predictions identified a significant reduction in the Pediatric Intensity Level of Therapy score beginning at 24 hours posttreatment through week 1 (p < 0.05). This divergence was also reflected in the Pediatric Logistic Organ Dysfunction score following the first week. The duration of intracranial pressure monitoring was 8.2 ± 1.3 days in the treated group and 15.6 ± 3.5 days (p = 0.03) in the time-matched control group. CONCLUSIONS: IV autologous bone marrow-derived mononuclear cell therapy is associated with lower treatment intensity required to manage intracranial pressure, associated severity of organ injury, and duration of neurointensive care following severe traumatic brain injury. This may corroborate preclinical data that autologous bone marrow-derived mononuclear cell therapy attenuates the effects of inflammation in the early post-traumatic brain injury period.
Asunto(s)
Trasplante de Médula Ósea/métodos , Lesiones Encefálicas/terapia , Presión Intracraneal , Monocitos/trasplante , Trasplante Autólogo/métodos , Índices de Gravedad del Trauma , Adolescente , Lesiones Encefálicas/fisiopatología , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Escala de Coma de Glasgow , Humanos , Infusiones Intravenosas , Masculino , Monocitos/citología , Estudios Retrospectivos , Factores de Tiempo , Resultado del TratamientoRESUMEN
BACKGROUND: Blood brain barrier (BBB) compromise is a key pathophysiological component of secondary traumatic brain injury characterized by edema and neuroinflammation in a previously immune-privileged environment. Current assays for BBB permeability are limited by working size, harsh extraction processes, suboptimal detection via absorbance, and wide excitation fluorescence spectra. In this study, we evaluate the feasibility of Alexa Fluor 680, a far-red dye bioconjugated to dextran, as an alternative assay to improve resolution and sensitivity. METHODS: Alexa Fluor was introduced intravenously on the day of sacrifice to three groups: sham, controlled cortical impact (CCI), and CCI treated with a cell based therapy known to reduce BBB permeability. The brains were sectioned coronally and imaged using an infrared laser scanner to generate intensity plot profiles as well as signal threshold images to distinguish regions with varying degrees of permeability. RESULTS: Linear plot profile analysis demonstrated greater signal intensity from CCI than treated rats at corresponding injury depths. Threshold analysis identified rims of signal at low + narrow threshold ranges. The integrated signals from a treatment group known to preserve the BBB were significantly less than the groups with CCI injury alone. There was no significant difference at high + wide signal intensity threshold ranges. CONCLUSIONS: Alexa Fluor 680 infrared photodetection and image analysis can aid in detecting differential degrees of BBB permeability after traumatic brain injury and maybe particularly useful in demonstrating BBB preservation of at-risk regions in response to therapeutic agents.
Asunto(s)
Barrera Hematoencefálica , Lesiones Encefálicas/fisiopatología , Permeabilidad Capilar , Dextranos , Colorantes Fluorescentes , Animales , Lesiones Encefálicas/terapia , Circulación Cerebrovascular/fisiología , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas/métodos , RatasRESUMEN
PURPOSE: Abnormal Notch signaling promotes cancer cell growth and tumor progression in various cancers. Targeting γ-secretase, a pivotal regulator in the Notch pathway, has yielded numerous γ-secretase inhibitors (GSIs) for clinical investigation in the last 2 decades. However, GSIs have demonstrated minimal success in clinical trials in part due to the lack of specific and precise tools to assess γ-secretase activity and its inhibition in vivo. EXPERIMENTAL DESIGN: We designed an imaging probe based on GSI Semagacestat structure and synthesized the radioiodine-labeled analogues [131I]- or [124I]-PN67 from corresponding trimethyl-tin precursors. Both membrane- and cell-based ligand-binding assays were performed using [131I]-PN67 to determine the binding affinity and specificity for γ-secretase in vitro. Moreover, we evaluated [124I]-PN67 by PET imaging in mammary tumor and glioblastoma mouse models. RESULTS: The probe was synthesized through iodo-destannylation using chloramine-T as an oxidant with a high labeling yield and efficiency. In vitro binding results demonstrate the high specificity of this probe and its ability for target replacement study by clinical GSIs. PET imaging studies demonstrated a significant (P < 0.05) increased in the uptake of [124I]-PN67 in tumors versus blocking or sham control groups across multiple mouse models, including 4T1 allograft, MMTV-PyMT breast cancer, and U87 glioblastoma allograft. Ex vivo biodistribution and autoradiography corroborate these results, indicating γ-secretase specific tumor accumulation of [124I]-PN67. CONCLUSIONS: [124I]-PN67 is a novel PET imaging agent that enables assessment of γ-secretase activity and target engagement of clinical GSIs.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide , Neoplasias de la Mama , Animales , Neoplasias de la Mama/patología , Femenino , Humanos , Radioisótopos de Yodo , Ratones , Tomografía de Emisión de Positrones , Receptores Notch/metabolismo , Distribución TisularRESUMEN
PURPOSE: Post-myocardial infarction heart failure is a major health concern with limited therapy. Molecular revascularisation utilising granulocyte-macrophage colony stimulating factor (GMCSF) mediated endothelial progenitor cell (EPC) upregulation and stromal cell derived factor-1α (SDF) mediated myocardial EPC chemokinesis, may prevent myocardial loss and adverse remodelling. Vasculogenesis, viability, and haemodynamic improvements following therapy were investigated. PROCEDURES: Lewis rats (n=91) underwent LAD ligation and received either intramyocardial SDF and subcutaneous GMCSF or saline injections at the time of infarction. Molecular and haemodynamic assessments were performed at pre-determined time points following ligation. FINDINGS: SDF/GMCSF therapy upregulated EPC density as shown by flow cytometry (0.12±0.02% vs. 0.06±0.01% circulating lymphocytes, p=0.005), 48hours following infarction. A marked increase in perfusion was evident eight weeks after therapy, utilising confocal angiography (5.02±1.7×10(-2)µm(3)blood/µm(3)myocardial tissue vs. 2.03±0.710(-2)µm(3)blood/µm(3)myocardial tissue, p=0.00004). Planimetric analysis demonstrated preservation of wall thickness (0.98±0.09mm vs. 0.67±0.06mm, p=0.003) and ventricular diameter (7.81±0.99mm vs. 9.41±1.1mm, p=0.03). Improved haemodynamic function was evidenced by echocardiography and PV analysis (ejection fraction: 56.4±18.1% vs. 25.3±15.6%, p=0.001; pre-load adjusted maximal power: 6.6±2.6mW/µl(2) vs. 2.7±1.4mW/µl(2), p=0.01). CONCLUSION: Neovasculogenic therapy with GMCSF-mediated EPC upregulation and SDF-mediated EPC chemokinesis maybe an effective therapy for infarct modulation and preservation of myocardial function following acute myocardial infarction.
Asunto(s)
Quimiocina CXCL12/uso terapéutico , Células Endoteliales/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Corazón/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Función Ventricular Izquierda/efectos de los fármacos , Animales , Apoptosis , Quimiocina CXCL12/farmacología , Ecocardiografía , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Ventrículos Cardíacos/anatomía & histología , Células Madre Hematopoyéticas/fisiología , Masculino , Modelos Animales , Miocardio/patología , Ratas , Ratas Endogámicas Lew , Regulación hacia ArribaRESUMEN
BACKGROUND: Heart failure is a global health concern. As a novel therapeutic strategy, the induction of endogenous myocardial regeneration was investigated by initiating cardiomyocyte mitosis by expressing the cell cycle regulator cyclin A2. METHODS AND RESULTS: Lewis rats underwent left anterior descending coronary artery ligation followed by peri-infarct intramyocardial delivery of adenoviral vector expressing cyclin A2 (n =32) or empty adeno-null (n =32). Cyclin A2 expression was characterized by Western Blot and immunohistochemistry. Six weeks after surgery, in vivo myocardial function was analyzed using an ascending aortic flow probe and pressure-volume catheter. DNA synthesis was analyzed by proliferating cell nuclear antigen (PCNA), Ki-67, and BrdU. Mitosis was analyzed by phosphohistone-H3 expression. Myofilament density and ventricular geometry were assessed. Cyclin A2 levels peaked at 2 weeks and tapered off by 4 weeks. Borderzone cardiomyocyte cell cycle activation was demonstrated by increased PCNA (40.1+/-2.6 versus 9.3+/-1.1; P<0.0001), Ki-67 (46.3+/-7.2 versus 20.4+/-6.0; P<0.0001), BrdU (44.2+/-13.7 versus 5.2+/-5.2; P<0.05), and phosphohistone-H3 (12.7+/-1.4 versus 0+/-0; P<0.0001) positive cells/hpf. Cyclin A2 hearts demonstrated increased borderzone myofilament density (39.8+/-1.1 versus 31.8+/-1.0 cells/hpf; P=0.0011). Borderzone wall thickness was greater in cyclin A2 hearts (1.7+/-0.4 versus 1.4+/-0.04 mm; P<0.0001). Cyclin A2 animals manifested improved hemodynamics: Pmax (70.6+/-8.9 versus 60.4+/-11.8 mm Hg; P=0.017), max dP/dt (3000+/-588 versus 2500+/-643 mm Hg/sec; P<0.05), preload adjusted maximal power (5.75+/-4.40 versus 2.75+/-0.98 mWatts/microL2; P<0.05), and cardiac output (26.8+/-3.7 versus 22.7+/-2.6 mL/min; P=0.004). CONCLUSIONS: A therapeutic strategy of cyclin A2 expression via gene transfer induced cardiomyocyte cell cycle activation yielded increased borderzone myofilament density and improved myocardial function. This approach of inducing endogenous myocardial regeneration provides proof-of-concept evidence that cyclin A2 may ultimately serve as an efficient, alternative therapy for heart failure.
Asunto(s)
Adenoviridae/genética , Ciclina A/fisiología , Virus Defectuosos/genética , Vectores Genéticos/uso terapéutico , Corazón/fisiología , Infarto del Miocardio/terapia , Regeneración/fisiología , Citoesqueleto de Actina/ultraestructura , Animales , Ciclo Celular/fisiología , División Celular , Ciclina A/genética , Ciclina A2 , Replicación del ADN , Vectores Genéticos/genética , Insuficiencia Cardíaca/prevención & control , Hemodinámica , Inyecciones , Masculino , Ratones , Infarto del Miocardio/fisiopatología , Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Distribución Aleatoria , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes de Fusión/fisiología , Transducción GenéticaRESUMEN
Apelin interacts with the APJ receptor to enhance inotropy. In heart failure, apelin-APJ coupling may provide a means of enhancing myocardial function. The alterations in apelin and APJ receptor concentrations with ischemic cardiomyopathy are poorly understood. We investigated the compensatory changes in endogenous apelin and APJ levels in the setting of ischemic cardiomyopathy.Male, Lewis rats underwent LAD ligation and progressed into heart failure over 6 weeks. Corresponding animals underwent sham thoracotomy as control. Six weeks after initial surgery, the animals underwent hemodynamic functional analysis in the presence of exogenous apelin-13 infusion and the hearts were explanted for western blot and enzyme immunoassay analysis. Western blot analysis of myocardial APJ concentration demonstrated increased APJ receptor protein levels with heart failure (1890750+/-133500 vs. 901600+/-143120 intensity units, n=8, p=0.00001). Total apelin protein levels increased with ischemic heart failure as demonstrated by enzyme immunoassay (12.0+/-4.6 vs. 1.0+/-1.2 ng/ml, n=5, p=0.006) and western blot (1579400+/-477733 vs. 943000+/-157600 intensity units, n=10, p=0.008). Infusion of apelin-13 significantly enhanced myocardial function in sham and failing hearts. We conclude that total myocardial apelin and APJ receptor levels increase in compensation for ischemic cardiomyopathy.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Isquemia Miocárdica/metabolismo , Miocardio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Apelina , Receptores de Apelina , Presión Sanguínea/efectos de los fármacos , Western Blotting , Gasto Cardíaco/efectos de los fármacos , Glicosilación/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/fisiopatología , Péptidos y Proteínas de Señalización Intercelular/administración & dosificación , Péptidos y Proteínas de Señalización Intercelular/farmacología , Masculino , Peso Molecular , Isquemia Miocárdica/fisiopatología , Miocardio/patología , Ratas , Ratas Endogámicas LewRESUMEN
The direct macrocycle synthesis of α-isocyano-ω-carboxylic acids via an Ugi multicomponent reaction is introduced. This multicomponent reaction (MCR) protocol differs by being especially short, convergent, and versatile, giving access to 12-22 membered rings.
Asunto(s)
Ácidos Carboxílicos/síntesis química , Compuestos Macrocíclicos/síntesis química , Ácidos Carboxílicos/química , Cristalografía por Rayos X , Compuestos Macrocíclicos/química , Conformación Molecular , Estructura Molecular , EstereoisomerismoRESUMEN
Basic bulky amines such as amantadine are well-characterized M2 channel blockers, useful for treating influenza. Herein we report our surprising findings that charge-neutral, bulky isocyanides exhibit activities similar to--or even higher than--that of amantadine. We also demonstrate that these isocyanides have potent growth inhibitory activity against the H5N1 virus. The -NH2 to -N≡C group replacement within current anti-influenza drugs was found to give compounds with high activities at low-micromolar concentrations. For example, a tenfold improvement in potency was observed for 1-isocyanoadamantane (27), with an EC50 value of 0.487â µm against amantadine-sensitive H5N1 virus as determined by both MTT and plaque-reduction assays, without showing cytotoxicity. Furthermore, the isocyanide analogues synthesized in this study did not inhibit the V27A or S31N mutant M2 ion channels, according to electrophysiology experiments, and did not exhibit activity against amantadine-resistant virus strains.
Asunto(s)
Antivirales/farmacología , Cianuros/farmacología , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Proteínas de la Matriz Viral/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/química , Cianuros/síntesis química , Cianuros/química , Perros , Relación Dosis-Respuesta a Droga , Subtipo H5N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Células de Riñón Canino Madin Darby/microbiología , Pruebas de Sensibilidad Microbiana , Relación Estructura-Actividad , Proteínas de la Matriz Viral/metabolismoRESUMEN
A three step synthesis of N-unsubstituted tetrazolo γ- and δ-lactams involving a key Ugi-4CR is presented. The compounds, otherwise difficult to access, are conveniently synthesized in overall good yields by our route. PDB analysis of the N-unsubstituted γ- and δ-lactam fragment reveals a strongly tri-directional hydrogen bond donor acceptor interaction with the amino acids of the binding sites.
RESUMEN
Traumatic injuries to the inferior vena cava continue to be associated with high mortality. The management of these injuries has been technically challenging and highly variable, often depending on factors that include the anatomic complexity and the severity of the insult. We report the first case in which a patient with massive exsanguination from an infrahepatic vena cava gunshot wound underwent successful repair with the aid of a novel variant active venovenous bypass circuit between the inferior vena cava and the pulmonary artery.
Asunto(s)
Puente Cardíaco Derecho/métodos , Vena Cava Inferior/lesiones , Vena Cava Inferior/cirugía , Heridas por Arma de Fuego/cirugía , Adolescente , Humanos , Hígado , MasculinoAsunto(s)
Anfotericina B/administración & dosificación , Complicaciones de la Diabetes/diagnóstico , Absceso Pulmonar/complicaciones , Mucormicosis/diagnóstico , Neumonía Estafilocócica/complicaciones , Esputo/microbiología , Adulto , Enfermedad Crónica , Femenino , Humanos , Pulmón/diagnóstico por imagen , Pulmón/patología , Mucormicosis/tratamiento farmacológico , Mucormicosis/cirugía , Tomografía Computarizada por Rayos X , Adulto JovenAsunto(s)
Intestino Delgado , Oclusión Vascular Mesentérica/diagnóstico , Protrombina/genética , Trombofilia/diagnóstico , Trombosis de la Vena/diagnóstico , Adulto , Marcadores Genéticos , Humanos , Intestino Delgado/irrigación sanguínea , Intestino Delgado/diagnóstico por imagen , Intestino Delgado/patología , Masculino , Oclusión Vascular Mesentérica/genética , Venas Mesentéricas , Mutación , Radiografía , Trombofilia/genética , Trombosis de la Vena/genéticaRESUMEN
OBJECTIVE: A significant number of patients have coronary artery disease that is not amenable to traditional revascularization. Prospective, randomized clinical trials have demonstrated therapeutic benefits with transmyocardial laser revascularization in this cohort. The molecular mechanisms underlying this therapy, however, are poorly understood. The focus of this study was evaluation of the proposed vasculogenic mechanisms involved in transmyocardial laser revascularization. METHODS: Male Yorkshire pigs (30-35 kg, n = 25) underwent left thoracotomy and placement of ameroid constrictors around the proximal left circumflex coronary artery. During the next 4 weeks, a well-defined region of myocardial ischemia developed, and the animals underwent a redo left thoracotomy. The animals were randomly assigned to sham treatment (thoracotomy only, control, n = 11) or transmyocardial laser revascularization of hibernating myocardium with a holmium:yttrium-aluminum-garnet laser (n = 14). After an additional 4 weeks, the animals underwent median sternotomy, echocardiographic analysis of wall motion, and hemodynamic analysis with an ascending aortic flow probe and pulmonary artery catheter. The hearts were explanted for molecular analysis. RESULTS: Molecular analysis demonstrated statistically significant increases in the proangiogenic proteins nuclear factor kappaB (42 +/- 27 intensity units vs 591 +/- 383 intensity units, P = .03) and angiopoietin 1 (0 +/- 0 intensity units vs 241 +/- 87 intensity units, P = .003) relative to sham control values with transmyocardial laser revascularization within the ischemic myocardium. There were also increases in vasculogenesis (18.8 +/- 8.7 vessels/high-power field vs 31.4 +/- 10.2 vessels/high-power field, P = .02), and perfusion (0.028 +/- 0.009 microm3 blood/microm3 tissue vs 0.044 +/- 0.004 microm3 blood/microm3 tissue, P = .01). Enhanced myocardial viability was demonstrated by increased myofilament density (40.7 +/- 8.5 cardiomyocytes/high-power field vs 50.8 +/- 7.5 cardiomyocytes/high-power field, P = .03). Regional myocardial function within the treated territory demonstrated augmented contractility. Global hemodynamic function was significantly improved relative to the control group with transmyocardial laser revascularization (cardiac output 2.1 +/- 0.2 L/min vs 2.7 +/- 0.2 L/min, P = .007, mixed venous oxygen saturation 64.7% +/- 3.6% vs 76.1% +/- 3.4%, P = .008). CONCLUSION: Transmyocardial laser revascularization with the holmium-YAG laser enhances perfusion, with resultant improvement in myocardial contractility.
Asunto(s)
Circulación Coronaria/fisiología , Terapia por Láser , Isquemia Miocárdica/cirugía , Revascularización Miocárdica/métodos , Animales , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Hemodinámica/fisiología , Láseres de Estado Sólido , Masculino , Contracción Miocárdica/fisiología , Neovascularización Fisiológica , Probabilidad , Distribución Aleatoria , Valores de Referencia , Sensibilidad y Especificidad , Porcinos , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: Heart failure therapies ranging from revascularization to remodeling to replacement are variably effective. Theoretically, endogenous repair via myocardial regeneration would be an ideal therapy. This study examined the ability to initiate regeneration by adenoviral-mediated expression of the cell cycle regulator cyclin A2. Our prior studies have demonstrated robust cyclin A2 transgene expression and marked antiphosphorylated histone H3 activity with this strategy, indicating the induction of cardiomyocyte mitosis. METHODS: Adult male, Lewis rats underwent left anterior descending coronary artery ligation followed by intramyocardial delivery of either cyclin A2 adenoviral vector (n = 8) or empty adeno-null vector as a control (n = 8) into the peri-infarct border zone. In vivo myocardial function was analyzed by echocardiography and invasive left ventricular pressure catheter at 6 weeks, when the animals are traditionally in heart failure. Hearts were explanted for immunoblotting and left ventricular geometric analysis. Cellular proliferation was assessed by proliferating cellular nuclear antigen expression. RESULTS: Cyclin A2 hearts exhibited improved left ventricular function as compared with controls including enhanced cardiac output (32 +/- 3.3 vs 26 +/- 5.0 mL/min, P < .05), stroke volume (0.16 +/- 0.04 vs 0.11 +/- 0.04 mL, P < .05), ejection fraction (72% +/- 7.4% vs 46.% +/- 8.5%, P < .05), fractional shortening (35% +/- 5.4% vs 19% +/- 4.3%, P < .002), maximum pressure (72 +/- 9.3 vs 61 +/- 2.9 mm Hg, P < .05), and end-systolic pressure (67 +/- 7.0 vs 55 +/- 7.0 mm Hg, P < .05). Enhanced myocardial preservation was demonstrated by enhanced left ventricular border zone wall thickness. Increased myocardial proliferation was evidenced by increased expression of proliferating cell nuclear antigen expression in cyclin A2-treated hearts. CONCLUSIONS: In failing hearts, targeted delivery of cyclin A2 improves hemodynamic function, as measured by echocardiography and pressure catheter analysis, preserves ventricular wall thickness, and may serve as an ideal myocardial regenerative therapy.
Asunto(s)
Cardiomiopatías/tratamiento farmacológico , Proteínas de Ciclo Celular/administración & dosificación , Ciclina A/administración & dosificación , Miocitos Cardíacos/fisiología , Regeneración/efectos de los fármacos , Adenoviridae , Animales , Cardiomiopatías/etiología , Ciclina A2 , Modelos Animales de Enfermedad , Vectores Genéticos , Inyecciones Intralesiones , Masculino , Infarto del Miocardio/complicaciones , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/etiología , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Ratas , Ratas Endogámicas Lew , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
Left ventricular dysfunction is a predictor of perioperative morbidity and mortality in on-pump coronary artery bypass grafting. Obligatory global myocardial ischemia and injury induced during crossclamping as well as adverse systemic effects of cardiopulmonary bypass may induce a disproportionately greater overall physiologic insult in patients with poor ventricular function. All patients undergoing nonemergency off-pump coronary artery bypass by a single surgeon during an 18-month period were retrospectively analyzed. Two groups with preoperative ejection fraction classified as poor (10%-35%; n = 31) or normal (55%-80%; n = 60) were compared. The mean ejection fractions were 26% +/- 1% and 63% +/- 1% respectively, p < 0.000001. In those with significant left ventricular dysfunction, there were 2.8 +/- 0.1 grafts per patient, time to extubation was 8.4 +/- 1.2 hours, and discharge was after 4.9 +/- 0.6 days. These results were statistically equivalent to those in the group with normal left ventricular function. There was no intraaortic balloon pump insertion or mortality in either group. This technique provides an effective means of safely revascularizing patients with significant left ventricular dysfunction, and it may provide a valuable alternative approach in patients with ischemic cardiomyopathy.