RESUMEN
Surgical excision and grafting of deep partial-thickness (DPT) and full-thickness (FT) burns is a cornerstone of wound care. The use of commercially available topical enzymatic agents has been limited due to slower and less complete eschar removal than surgical excision. Using a porcine model of DPT and FT burns, we compared the eschar removal efficacy of a bromelain-enriched enzymatic agent derived from the stems of pineapple plants and a commercially available collagenase. We created 40 DPT and 40 FT burns on four anesthetized Yorkshire pigs. Eschar removal was initiated 24 hours later. Two pigs each were randomly assigned to collagenase or the bromelain-enriched agent. The bromelain-enriched agent was applied topically once for 4 hours followed by a 2-hour soaking. The collagenase was applied topically daily until complete removal of eschar or for up to 14 days. All bromelain-enriched treated FT burns underwent complete removal of the eschar after a single application while none of the collagenase-treated FT burns underwent complete removal of the eschar even after 14 days of treatment. All bromelain-enriched treated DPT burns had complete eschar removal after the single application. None of the collagenase-treated DPT burns experienced complete removal of eschar after 10 days; by day 14, 35% had complete eschar removal, 30% had >50% eschar removed, and 35% had <50% eschar removed. We conclude that eschar removal is quicker and more complete with the bromelain-enriched compared with collagenase debriding agent.
Asunto(s)
Quemaduras , Cicatrización de Heridas , Animales , Bromelaínas/farmacología , Bromelaínas/uso terapéutico , Quemaduras/tratamiento farmacológico , Quemaduras/cirugía , Colagenasas/farmacología , Desbridamiento , PorcinosRESUMEN
Von Hippel-Lindau (VHL) disease is a tumor predisposition syndrome caused by mutations in the VHL gene that presents with visceral neoplasms and growths, including clear cell renal cell carcinoma, and central nervous system manifestations, such as hemangioblastomas of the brain and spine. The pathophysiology involves dysregulation of oxygen sensing caused by the inability to degrade HIFα, leading to the overactivation of hypoxic pathways. Hemangioblastomas are the most common tumors in patients with VHL and cause significant morbidity. Until recently, there were no systemic therapies available for patients that could effectively reduce the size of these lesions. Belzutifan, the first approved HIF-2α inhibitor, has demonstrated benefit in VHL-associated tumors, with a 30% response rate in hemangioblastomas and ~30%-50% reduction in their sizes over the course of treatment. Anemia is the most prominent adverse effect, affecting 76%-90% of participants and sometimes requiring dose reduction or transfusion. Other significant adverse events include hypoxia and fatigue. Overall, belzutifan is well tolerated; however, long-term data on dosing regimens, safety, and fertility are not yet available. Belzutifan holds promise for the treatment of neurological manifestations of VHL and its utility may influence the clinical management paradigms for this patient population.
Asunto(s)
Hemangioblastoma , Neoplasias Renales , Enfermedad de von Hippel-Lindau , Humanos , Enfermedad de von Hippel-Lindau/genética , Hemangioblastoma/tratamiento farmacológico , Hemangioblastoma/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
Macrophage-like cells (MLCs) are potential inflammatory biomarkers. We previously showed that MLCs are increased in proliferative diabetic retinopathy (PDR) eyes. Vision-threatening diabetic retinopathy (VTDR) includes PDR, severe non-PDR (NPDR), and diabetic macular edema (DME). No prior data exist on MLCs in eyes with severe NPDR or DME. This prospective, cross-sectional optical coherence tomography-angiography (OCT-A) imaging study included 40 eyes of 37 participants who had NPDR classified as non-VTDR (n = 18) or VTDR (n = 22). Repeated OCT-A images were registered, averaged, and used to quantify the main outcome measures: MLC density and percent area. MLC density and percent area were correlated with clinical characteristics, NPDR stage, presence of DME, and OCT central subfield thickness (CST). In VTDR eyes, MLC density (2.6-fold, p < 0.001) and MLC percent area (2.5-fold, p < 0.01) were increased compared with non-VTDR eyes. Multiple linear regression analysis between MLC metrics and clinical characteristics found that MLC density was positively correlated with worse NPDR severity (p = 0.023) and higher CST values (p = 0.010), while MLC percent area was only positively associated with increased CST values (p = 0.006). MLCs are increased in patients with VTDR. Macular edema is the most strongly associated factor with increased MLC numbers in NPDR eyes.
RESUMEN
CRISPR/Cas9-mediated genome editing provides potential for therapeutic development. Efficacy and long-term safety represent major concerns that remain to be adequately addressed in preclinical studies. Here we show that CRISPR/Cas9-mediated genome editing in two distinct SOD1-amyotrophic lateral sclerosis (ALS) transgenic mouse models prevented the development of ALS-like disease and pathology. The disease-linked transgene was effectively edited, with rare off-target editing events. We observed frequent large DNA deletions, ranging from a few hundred to several thousand base pairs. We determined that these large deletions were mediated by proximate identical sequences in Alu elements. No evidence of other diseases was observed beyond 2 years of age in these genome edited mice. Our data provide preclinical evidence of the efficacy and long-term safety of the CRISPR/Cas9 therapeutic approach. Moreover, the molecular mechanism of proximate identical sequences-mediated recombination provides mechanistic information to optimize therapeutic targeting design, and to avoid or minimize unintended and potentially deleterious recombination events.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Sistemas CRISPR-Cas/genética , Edición Génica/estadística & datos numéricos , Superóxido Dismutasa-1/genética , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Superóxido Dismutasa-1/química , Superóxido Dismutasa-1/metabolismoRESUMEN
Whether the depth and healing of scalds and contact burns are similar is controversial. Due to water's greater heat capacity, we hypothesized that when exposed to similar temperatures and durations of exposure, burns caused by hot water would be deeper than those caused by contact with hot metal. Forty standardized burns were created in two anesthetized female domestic pigs using a brass bar or circulating heated water. In one pig, the temperature was kept constant (95°C) while the duration of exposure varied (5, 10, 15 seconds) In the second pig, the exposure time was kept constant (10 seconds) while the temperature of exposure varied (70°C, 80°C, 98°C). Periodic punch biopsies were taken to determine burn depth immediately after injury, percentage burns reepithelialized within 21 days, and depth of scar at 28 days. The analysis was performed using analysis of variance. When the temperature was held constant, duration of exposure (5, 10, and 15 seconds) was associated with scar depth (2.1 vs 3.8 vs 5.0 mm, respectively, P = 0.001) but not with burn depth (2.0 vs 2.2 vs 2.3 mm, respectively, P = 0.10). When exposure duration was held constant, temperature (70°C, 80°C, 98°C) was associated with scar depth (0.6 vs 1.7 vs 3.6, P < 0.001) but not with burn depth (1.2 vs 1.5 vs 1.7 mm, respectively, P = 0.21). Burn depths were greater for scald than contact burns although not significantly greater. After controlling for temperature, the difference in scar depth between scalds and contact burns was statistically significant (marginal means 3.0 for contact burns, 4.3 for scalds, P = 0.008). We conclude that burns created in swine with circulating hot water result in deeper scars than those created by contact with a brass bar when controlling for temperature and duration of exposure.
Asunto(s)
Quemaduras/diagnóstico , Cicatriz/diagnóstico , Repitelización/fisiología , Piel/lesiones , Cicatrización de Heridas/fisiología , Animales , Biopsia , Quemaduras/complicaciones , Cicatriz/etiología , Modelos Animales de Enfermedad , Femenino , Calor/efectos adversos , Estudios Prospectivos , Piel/patología , Porcinos , Índices de Gravedad del TraumaRESUMEN
BACKGROUND: Burn injury progression in the secondary zone of ischemia is common leading to delayed healing and increased scarring. We hypothesized that a topical surfactant, would reduce burn injury progression in a validated rat comb burn model compared with topical antibiotic ointment. METHODS: We created 40 comb burns on 20 rats which were randomized to daily topical application of the surfactant or a triple antibiotic ointment. The comb burns consisted of 4 full thickness burns with 3 unburned interspaces between the 4 burns. These unburned interspaces represented the zone of ischemia, and when left untreated, generally progress to full thickness necrosis within several days. Comb burns were assessed daily for the presence of gross necrosis of the interspaces. At 7 days the comb burns were excised and blindly evaluated for the presence of histological evidence of necrosis. The study had 80% power to detect a 25% difference in the percentages of necrotic interspaces on day 7. RESULTS: There were no differences in the percentages of histologically necrotic interspaces at 7 days in burns treated with the surfactant or antibiotic ointment (85% [95%CI, 74 to 92] vs. 75% [95%CI, 63 to 84]; mean difference 10% [95%CI -4 to 24]). There were also no between group differences in the percentages of grossly necrotic interspaces on any of the seven days of the experiment. The surfactant remained intact and adherent while the antibiotic had been absorbed at each daily dressing change. CONCLUSIONS: A topical surfactant did not reduce injury progression in the rat comb burn model when compared with antibiotic ointment. The surfactant was more durable than the antibiotic ointment.
Asunto(s)
Antibacterianos , Quemaduras , Pomadas , Tensoactivos , Animales , Antibacterianos/administración & dosificación , Antibacterianos/uso terapéutico , Quemaduras/tratamiento farmacológico , Modelos Animales de Enfermedad , Isquemia , Necrosis , Ratas , Tensoactivos/administración & dosificación , Tensoactivos/uso terapéuticoRESUMEN
Purpose: The objective of this study was to understand the molecular and physiologic mechanisms behind the lens cataract differences in Aquaporin 0-knockout-Heterozygous (AQP0-Htz) mice developed in C57 and FVB (lacks beaded filaments [BFs]) strains. Methods: Lens transparency was studied using dark field light microscopy. Water permeability (Pf) was measured in fiber cell membrane vesicles. Western blotting/immunostaining was performed to verify expression of BF proteins and connexins. Microelectrode-based intact lens intracellular impedance was measured to determine gap junction (GJ) coupling resistance. Lens intracellular hydrostatic pressure (HP) was determined using a microelectrode/manometer system. Results: Lens opacity and spherical aberration were more distinct in AQP0-Htz lenses from FVB than C57 strains. In either background, compared to wild type (WT), AQP0-Htz lenses showed decreased Pf (approximately 50%), which was restored by transgenic expression of AQP1 (TgAQP1/AQP0-Htz), but the opacities and differences between FVB and C57 persisted. Western blotting revealed no change in connexin expression levels. However, in C57 AQP0-Htz and TgAQP1/AQP0-Htz lenses, GJ coupling resistance decreased approximately 2.8-fold and the HP gradient decreased approximately 1.9-fold. Increased Pf in TgAQP1/AQP0-Htz did not alter GJ coupling resistance or HP. Conclusions: In C57 AQP0-Htz lenses, GJ coupling resistance decreased. HP reduction was smaller than the coupling resistance reduction, a reflection of an increase in fluid circulation, which is one reason for the less severe cataract in C57 than FVB. Overall, our results suggest that AQP0 modulates GJs in the presence of BF proteins to maintain lens transparency and homeostasis.
Asunto(s)
Acuaporina 1/genética , Catarata/genética , Proteínas del Ojo/genética , Regulación de la Expresión Génica , Proteínas de Filamentos Intermediarios/genética , Cristalino/metabolismo , ARN/genética , Animales , Acuaporina 1/biosíntesis , Western Blotting , Catarata/metabolismo , Catarata/patología , Modelos Animales de Enfermedad , Impedancia Eléctrica , Proteínas del Ojo/biosíntesis , Uniones Comunicantes/genética , Uniones Comunicantes/metabolismo , Genotipo , Heterocigoto , Proteínas de Filamentos Intermediarios/biosíntesis , Cristalino/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroelectrodosRESUMEN
Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Terapia Genética/métodos , Oligonucleótidos Antisentido/farmacología , Animales , Células COS , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Repeticiones de Microsatélite , Empalme del ARN , Expansión de Repetición de TrinucleótidoRESUMEN
PURPOSE: The goals of the study were to elucidate the immune mechanisms that contribute to desirable complete remissions of murine colon tumors treated with single radiation dose of 30 Gy. This dose is at the upper end of the ablative range used clinically to treat advanced or metastatic colorectal, liver, and non-small cell lung tumors. EXPERIMENTAL DESIGN: Changes in the tumor immune microenvironment of single tumor nodules exposed to radiation were studied using 21-day (>1 cm in diameter) CT26 and MC38 colon tumors. These are well-characterized weakly immunogenic tumors. RESULTS: We found that the high-dose radiation transformed the immunosuppressive tumor microenvironment resulting in an intense CD8(+) T-cell tumor infiltrate, and a loss of myeloid-derived suppressor cells (MDSC). The change was dependent on antigen cross-presenting CD8(+) dendritic cells, secretion of IFNγ, and CD4(+)T cells expressing CD40L. Antitumor CD8(+) T cells entered tumors shortly after radiotherapy, reversed MDSC infiltration, and mediated durable remissions in an IFNγ-dependent manner. Interestingly, extended fractionated radiation regimen did not result in robust CD8(+) T-cell infiltration. CONCLUSIONS: For immunologically sensitive tumors, these results indicate that remissions induced by a short course of high-dose radiotherapy depend on the development of antitumor immunity that is reflected by the nature and kinetics of changes induced in the tumor cell microenvironment. These results suggest that systematic examination of the tumor immune microenvironment may help in optimizing the radiation regimen used to treat tumors by adding a robust immune response.