RESUMEN
The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.
Asunto(s)
Proteínas de Unión al ADN/química , Transposasas/química , Secuencia de Bases , ADN/química , ADN/metabolismo , Elementos Transponibles de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Simulación del Acoplamiento Molecular , Mutación , Unión Proteica , Dominios Proteicos , Transposasas/genética , Transposasas/metabolismo , Zinc/química , Dedos de ZincRESUMEN
OBJECTIVE: To evaluate the performance of a 2-tiered newborn screening method for mucopolysaccharidosis type I (MPS I) in North Carolina. STUDY DESIGN: The screening algorithm included a flow injection analysis-tandem mass spectrometry assay as a first-tier screening method to measure α-L-iduronidase (IDUA) enzyme activity and Sanger sequencing of the IDUA gene on dried blood spots as a second-tier assay. The screening algorithm was revised to incorporate the Collaborative Laboratory Integrated Reports, an analytical interpretive tool, to reduce the false-positive rate. A medical history, physical examination, IDUA activity, and urinary glycosaminoglycan (GAG) analysis were obtained on all screen-positive infants. RESULTS: A total of 62 734 specimens were screened with 54 screen-positive samples using a cut-off of 15% of daily mean IDUA activity. The implementation of Collaborative Laboratory Integrated Reports reduced the number of specimens that screened positive to 19 infants. Of the infants identified as screen-positive, 1 had elevated urinary GAGs and a homozygous pathogenic variant associated with the severe form of MPS I. All other screen-positive infants had normal urinary GAG analysis; 13 newborns had pseudodeficiency alleles, 3 newborns had variants of unknown significance, and 2 had heterozygous pathogenic variants. CONCLUSIONS: An infant with severe MPS I was identified and referred for a hematopoietic stem cell transplant. Newborn IDUA enzyme deficiency is common in North Carolina, but most are due to pseudodeficiency alleles in infants with normal urinary GAG analysis and no evidence of disease. The pilot study confirmed the need for second-tier testing to reduce the follow-up burden.
Asunto(s)
Mucopolisacaridosis I/diagnóstico , Tamizaje Neonatal , Algoritmos , Dermatán Sulfato/orina , Pruebas Genéticas , Variación Genética , Glicosaminoglicanos/orina , Heparitina Sulfato/orina , Humanos , Iduronidasa/sangre , Iduronidasa/genética , Recién Nacido , Mucopolisacaridosis I/genética , North Carolina , Derivación y Consulta/estadística & datos numéricos , Análisis de Secuencia , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Newborn screening (NBS) occupies a unique space at the intersection of translational science and public health. As the only truly population-based public health program in the United States, NBS offers the promise of making the successes of translational medicine available to every infant with a rare disorder that is difficult to diagnose clinically, but for which strong evidence indicates that presymptomatic treatment will substantially improve outcomes. Realistic NBS policy requires data, but rare disorders face a special challenge: Screening cannot be done without supportive data, but adequate data cannot be collected in the absence of large-scale screening. The magnitude and scale of research to provide this expanse of data require working with public health programs, but most do not have the resources or mandate to conduct research. METHODS: To address this gap, we have established Early Check, a research program in partnership with a state NBS program. Early Check provides the infrastructure needed to identify conditions for which there have been significant advances in treatment potential, but require a large-scale, population-based study to test benefits and risks, demonstrate feasibility, and inform NBS policy. DISCUSSION: Our goal is to prove the benefits of a program that can, when compared with current models, accelerate understanding of diseases and treatments, reduce the time needed to consider inclusion of appropriate conditions in the standard NBS panel, and accelerate future research on new NBS conditions, including clinical trials for investigational interventions. TRIAL REGISTRATION: Clinicaltrials.gov registration # NCT03655223 . Registered on August 31, 2018.
Asunto(s)
Síndrome del Cromosoma X Frágil/diagnóstico , Atrofia Muscular Espinal/diagnóstico , Tamizaje Neonatal , Salud Pública , Investigación Biomédica Traslacional , Diagnóstico Precoz , Femenino , Estudios de Seguimiento , Síndrome del Cromosoma X Frágil/epidemiología , Política de Salud , Humanos , Recién Nacido , Consentimiento Informado , Internet , Colaboración Intersectorial , Masculino , Atrofia Muscular Espinal/epidemiología , North Carolina/epidemiología , Evaluación de Resultado en la Atención de Salud/métodos , Selección de Paciente , Evaluación de Programas y Proyectos de Salud , Estudios Prospectivos , Grupos de AutoayudaRESUMEN
This commentary discusses the importance of conducting newborn screening pilot studies in North Carolina and the lessons learned from performing three pilots for severe combined immunodeficiency (SCID), mucopolysaccharidosis type I (MPS I), and X-linked adrenoleukodystrophy (X-ALD).
Asunto(s)
Adrenoleucodistrofia/diagnóstico , Mucopolisacaridosis I/diagnóstico , Tamizaje Neonatal , Inmunodeficiencia Combinada Grave/diagnóstico , Humanos , Recién Nacido , North Carolina , Proyectos PilotoRESUMEN
BACKGROUND AND AIMS: The aim of this study was to compare endoscopy and pathology sizing in a large population-based series of colorectal adenomas and to evaluate the implications for patient stratification into surveillance colonoscopy. METHODS: Endoscopy and pathology sizes available from intact adenomas removed at colonoscopies performed as part of the Northern Ireland Bowel Cancer Screening Programme, from 2010 to 2015, were included in this study. Chi-squared tests were applied to compare size categories in relation to clinicopathologic parameters and colonoscopy surveillance strata according to current American Gastroenterology Association and British Society of Gastroenterology guidelines. RESULTS: A total of 2521 adenomas from 1467 individuals were included. There was a trend toward larger endoscopy than pathology sizing in 4 of the 5 study centers, but overall sizing concordance was good. Significantly greater clustering with sizing to the nearest 5 mm was evident in endoscopy versus pathology sizing (30% vs 19%, P < .001), which may result in lower accuracy. Applying a 10-mm cut-off relevant to guidelines on risk stratification, 7.3% of all adenomas and 28.3% of those 8 to 12 mm in size had discordant endoscopy and pathology size categorization. Depending on which guidelines are applied, 4.8% to 9.1% of individuals had differing risk stratification for surveillance recommendations, with the use of pathology sizing resulting in marginally fewer recommended surveillance colonoscopies. CONCLUSIONS: Choice of pathology or endoscopy approaches to determine adenoma size will potentially influence surveillance colonoscopy follow-up in 4.8% to 9.1% of individuals. Pathology sizing appears more accurate than endoscopy sizing, and preferential use of pathology size would result in a small, but clinically important, decreased burden on surveillance colonoscopy demand. Careful endoscopy sizing is required for adenomas removed piecemeal.
Asunto(s)
Adenoma/patología , Colonoscopía , Neoplasias Colorrectales/patología , Anciano , Detección Precoz del Cáncer , Femenino , Humanos , Masculino , Persona de Mediana Edad , Irlanda del Norte , Carga TumoralRESUMEN
BACKGROUND: Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn blood spot screening (NBS) to detect severe combined immunodeficiency (SCID) has been implemented in public health laboratories in the last 5 years. SCID detection is based on real-time PCR assays to measure T-cell receptor excision circles (TREC), a byproduct of T-cell development. We modified a multiplexed real-time PCR TREC assay to simultaneously determine the presence or absence of the SMN1 gene from a dried blood spot (DBS) punch in a single reaction well. METHOD: An SMN1 assay using a locked nucleic acid probe was initially developed with cell culture and umbilical cord blood (UCB) DNA extracts, and then integrated into the TREC assay. DBS punches were placed in 96-well arrays, washed, and amplified directly using reagents specific for TREC, a reference gene [ribonuclease P/MRP 30kDa subunit (RPP30)], and the SMN1 gene. The assay was tested on DBS made from UCB units and from peripheral blood samples of SMA-affected individuals and their family members. RESULTS: DBS made from SMA-affected individuals showed no SMN1-specific amplification, whereas DBS made from all unaffected carriers and UCB showed SMN1 amplification above a well-defined threshold. TREC and RPP30 content in all DBS were within the age-adjusted expected range. CONCLUSIONS: SMA caused by the absence of SMN1 can be detected from the same DBS punch used to screen newborns for SCID.
Asunto(s)
ADN/genética , Pruebas con Sangre Seca/métodos , Atrofia Muscular Espinal/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptores de Antígenos de Linfocitos T/genética , Inmunodeficiencia Combinada Grave/diagnóstico , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Adolescente , Adulto , Niño , Preescolar , ADN/sangre , Pruebas Genéticas/métodos , Humanos , Lactante , Recién Nacido , Persona de Mediana Edad , Atrofia Muscular Espinal/sangre , Atrofia Muscular Espinal/genética , Inmunodeficiencia Combinada Grave/sangre , Inmunodeficiencia Combinada Grave/genética , Proteína 1 para la Supervivencia de la Neurona Motora/sangre , Adulto JovenRESUMEN
NEW FINDINGS: What is the central question of this study? Hyperoxia blunts hypoglycaemia counterregulation in healthy adults. We hypothesized that this effect is mediated by the carotid bodies and that: (i) hyperoxia would have no effect on hypoglycaemia counterregulation in carotid body-resected patients; and (ii) carotid body-resected patients would exhibit an impaired counterregulatory response to hypoglycaemia. What is the main finding and its importance? Our data indicate that the effect of hyperoxia on hypoglycaemic counterregulation is mediated by the carotid bodies. However, a relatively normal counterregulatory response to hypoglycaemia in carotid body-resected patients highlights: (i) the potential for long-term adaptations after carotid body resection; and (ii) the importance of redundant mechanisms in mediating hypoglycaemia counterregulation. Hyperoxia reduces hypoglycaemia counterregulation in healthy adults. We hypothesized that this effect is mediated by the carotid bodies and that: (i) hyperoxia would have no effect on hypoglycaemia counterregulation in patients with bilateral carotid body resection; and (ii) carotid body-resected patients would exhibit an impaired counterregulatory response to hypoglycaemia. Five patients (three male and two female) with bilateral carotid body resection for glomus tumours underwent two 180 min hyperinsulinaemic, hypoglycaemic (â¼ 3.3 mmol l(-1)) clamps separated by a minimum of 1 week and randomized to either normoxia (21% fractional inspired O2 ) or hyperoxia (100% fractional inspired O2). Ten healthy adults (seven male and three female) served as control subjects. Hypoglycaemia counterregulation in carotid body-resected patients was not significantly altered by hyperoxia (area under the curve expressed as a percentage of the normoxic response: glucose infusion rate, 111 ± 10%; cortisol, 94 ± 6%; glucagon, 107 ± 7%; growth hormone, 92 ± 10%; adrenaline, 89 ± 26%; noradrenaline, 79 ± 15%; main effect of condition, P > 0.05). This is in contrast to previously published results from healthy adults. However, the counterregulatory responses to hypoglycaemia during normoxia were not impaired in carotid body-resected patients when compared with control subjects (main effect of group, P > 0.05). Our data provide further corroborative evidence that the effect of hyperoxia on hypoglycaemic counterregulation is mediated by the carotid bodies. However, relatively normal counterregulatory responses to hypoglycaemia in carotid body-resected patients highlight the importance of redundant mechanisms in mediating hypoglycaemia counterregulation.
Asunto(s)
Tumor del Cuerpo Carotídeo/cirugía , Cuerpo Carotídeo/cirugía , Tumor Glómico/cirugía , Hiperoxia/fisiopatología , Hipoglucemia/fisiopatología , Adaptación Fisiológica , Adulto , Biomarcadores/sangre , Glucemia/metabolismo , Cuerpo Carotídeo/fisiopatología , Tumor del Cuerpo Carotídeo/fisiopatología , Femenino , Tumor Glómico/fisiopatología , Humanos , Hiperoxia/sangre , Hipoglucemia/sangre , Insulina/sangre , Masculino , Persona de Mediana Edad , Minnesota , Factores de Tiempo , Adulto JovenRESUMEN
We have previously shown that non-selective cyclo-oxygenase inhibition, via indomethacin, unfavourably increased central blood pressure in older adults, with little effect in young adults. In addition, the vasoactive prostaglandins have been shown to contribute to both peripheral vasodilator responses and large artery function; however, there is little information available in older adults and conflicting reports in young adults on the extent to which resistance vessel function is influenced by indomethacin. Thus, we tested the hypothesis that cyclo-oxygenase inhibition using indomethacin would attenuate forearm vascular conductance during reactive hyperaemia in older adults compared with young adults. Forearm blood flow responses to 5 min of forearm ischaemia were measured in 26 healthy adults (13 young, 25 ± 5 years old; and 13 older, 65 ± 6 years old), using venous occlusion plethysmography before and after indomethacin. Baseline forearm blood flow and vascular conductance were not different between groups during either trial, and there were no age-related differences prior to cyclo-oxygenase inhibition. Peak forearm vascular conductance and blood flow were similar between groups before indomethacin, but lower in older adults after indomethacin compared with young adults (27 ± 4 versus 41 ± 4 ml (100 ml)(-1) min(-1) (100 mmHg)(-1), P = 0.02; and 23 ± 3 versus 33 ± 3 ml (100 ml)(-1) min(-1), P = 0.02, respectively). These results, in conjunction with our previous findings in large arteries, suggest that ageing alters the effect of cyclo-oxygenase inhibition on vascular responses, and specifically, the resistance vessel responses underlying reactive hyperaemia.
Asunto(s)
Envejecimiento/fisiología , Inhibidores de la Ciclooxigenasa/farmacología , Antebrazo/fisiología , Hiperemia/fisiopatología , Indometacina/farmacología , Flujo Sanguíneo Regional/fisiología , Adolescente , Adulto , Anciano , Envejecimiento/efectos de los fármacos , Femenino , Antebrazo/irrigación sanguínea , Estado de Salud , Humanos , Masculino , Persona de Mediana Edad , Flujo Sanguíneo Regional/efectos de los fármacos , Adulto JovenRESUMEN
Activation of the carotid body chemoreceptors with hypoxia alters baroreceptor-mediated responses. We aimed to examine whether this relationship can be translated to other chemoreceptor stimuli (i.e. hypoglycaemia) by testing the following hypotheses: (i) activation of the carotid body chemoreceptors with hypoglycaemia would reduce spontaneous cardiac baroreflex sensitivity (sCBRS) in healthy humans; and (ii) desensitization of the carotid chemoreceptors with hyperoxia would restore sCBRS to baseline levels during hypoglycaemia. Ten young healthy adults completed two 180 min hyperinsulinaemic [2 mU (kg fat-free mass)(-1) min(-1)], hypoglycaemic (â¼ 3.2 µmol ml(-1)) clamps, separated by at least 1 week and randomized to normoxia (arterial partial pressure of O2, 122 ± 10 mmHg) or hyperoxia (arterial partial pressure of O2, 424 ± 123 mmHg; to blunt activation of the carotid body glomus cells). Changes in heart rate, blood pressure, plasma catecholamines, heart rate variability (HRV) and sCBRS were assessed. During hypoglycaemia, HRV and sCBRS were reduced (P < 0.05) and the baroreflex working range was shifted to higher heart rates. When hyperoxia was superimposed on hypoglycaemia, there was a greater reduction in blood pressure and a blunted rise in heart rate when compared with normoxic conditions (P < 0.05); however, there was no detectable effect of hyperoxia on sCBRS or HRV during hypoglycaemia (P > 0.05). In summary, hypoglycaemia-mediated changes in HRV and sCBRS cannot be attributed exclusively to the carotid chemoreceptors; however, the chemoreceptors appear to play a role in resetting the baroreflex working range during hypoglycaemia.
Asunto(s)
Barorreflejo , Presión Sanguínea , Cuerpo Carotídeo/fisiopatología , Hipoglucemia/fisiopatología , Adulto , Glucemia/metabolismo , Cuerpo Carotídeo/metabolismo , Catecolaminas/sangre , Femenino , Técnica de Clampeo de la Glucosa , Frecuencia Cardíaca , Humanos , Hiperoxia/fisiopatología , Hipoglucemia/sangre , Insulina/sangre , Masculino , Respiración , Adulto JovenRESUMEN
We have recently shown that effective cytokine gene therapy of solid tumors in HLA-A2 transgenic (HHD) mice lacking murine MHC class I molecule expression results in the generation of HLA-A2-restricted CD8(+) T effector cells selectively recognizing tumor blood vessel-associated pericytes and/or vascular endothelial cells. Using an HHD model in which HLA-A2(neg) tumor (MC38 colon carcinoma or B16 melanoma) cells are not recognized by the CD8(+) T cell repertoire, we now show that vaccines on the basis of tumor-associated blood vessel Ags (TBVA) elicit protective Tc1-dependent immunity capable of mediating tumor regression or extending overall survival. Vaccine efficacy was not observed if (HLA-A2(neg)) wild-type C57BL/6 mice were instead used as recipient animals. In the HHD model, effective vaccination resulted in profound infiltration of tumor lesions by CD8(+) (but not CD4(+)) T cells, in a coordinate reduction of CD31(+) blood vessels in the tumor microenvironment, and in the "spreading" of CD8(+) T cell responses to alternate TBVA that were not intrinsic to the vaccine. Protective Tc1-mediated immunity was durable and directly recognized pericytes and/or vascular endothelial cells flow-sorted from tumor tissue but not from tumor-uninvolved normal kidneys harvested from these same animals. Strikingly, the depletion of CD8(+), but not CD4(+), T cells at late time points after effective therapy frequently resulted in the recurrence of disease at the site of the regressed primary lesion. This suggests that the vaccine-induced anti-TBVA T cell repertoire can mediate the clinically preferred outcomes of either effectively eradicating tumors or policing a state of (occult) tumor dormancy.
Asunto(s)
Vasos Sanguíneos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Células Endoteliales/inmunología , Antígeno HLA-A2/inmunología , Neoplasias Experimentales/inmunología , Pericitos/inmunología , Animales , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/administración & dosificación , Línea Celular Tumoral , Genes MHC Clase I , Antígeno HLA-A2/genética , Activación de Linfocitos/inmunología , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Experimentales/irrigación sanguínea , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Tumor blood vessels are frequently inefficient in their design and function, leading to high interstitial fluid pressure, hypoxia, and acidosis in the tumor microenvironment (TME), rendering tumors refractory to the delivery of chemotherapeutic agents and immune effector cells. Here we identified the NOTCH antagonist delta-like 1 homologue (DLK1) as a vascular pericyte-associated antigen expressed in renal cell carcinomas (RCC), but not in normal kidney tissues in mice and humans. Vaccination of mice bearing established RCC against DLK1 led to immune-mediated elimination of DLK1(+) pericytes and to blood vessel normalization (i.e., decreased vascular permeability and intratumoral hypoxia) in the TME, in association with tumor growth suppression. After therapeutic vaccination, tumors displayed increased prevalence of activated VCAM1(+)CD31(+) vascular endothelial cells (VECs) and CXCL10, a type-1 T cell recruiting chemokine, in concert with increased levels of type-1 CD8(+) tumor-infiltrating lymphocytes (TIL). Vaccination against DLK1 also yielded (i) dramatic reductions in Jarid1B(+), CD133(+), and CD44(+) (hypoxia-responsive) stromal cell populations, (ii) enhanced tumor cell apoptosis, and (iii) increased NOTCH signaling in the TME. Coadministration of a γ-secretase inhibitor (N-[N-(3,5-Difluorophenacetyl-l-alanyl)]-(S)-phenylglycine t-butyl ester (DAPT)) that interferes with canonical NOTCH signaling resulted in the partial loss of therapeutic benefits associated with lentivirus encoding full-length murine (lvDLK1)-based vaccination.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/terapia , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/inmunología , Animales , Apoptosis , Linfocitos T CD8-positivos/inmunología , Proteínas de Unión al Calcio , Vacunas contra el Cáncer/inmunología , Permeabilidad Capilar , Carcinoma de Células Renales/genética , Hipoxia de la Célula , Línea Celular Tumoral , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Femenino , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lentivirus/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Pericitos/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Microambiente Tumoral , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/uso terapéuticoRESUMEN
PURPOSE: We hypothesized that adults with type 1 diabetes mellitus (T1DM) would exhibit impaired heart rate variability (HRV), QT interval, T-wave amplitude, and baroreflex sensitivity (BRS) when compared with healthy controls. In addition, we hypothesized that acute hypoglycemia would result in further adverse changes in measures of autonomic and cardiovascular function. METHODS: A single 180-min hyperinsulinemic (2 mU/kg TBW/min), hypoglycemic (~3.3 umol/mL) clamp was completed in 10 healthy adults and 13 adults with T1DM. Counterregulatory hormones were assessed and measures of heart rate (electrocardiogram) and blood pressure (intra-arterial catheter or finger photoplethysmography) were analyzed at baseline and during the hypoglycemic clamp for measures of HRV, QT interval, T-wave amplitude, and spontaneous cardiac BRS (sCBRS). RESULTS: Baseline measures of HRV, sCBRS, and T-wave amplitude were blunted in adults with T1DM when compared with healthy controls. Hypoglycemia resulted in significant reductions in HRV, sCBRS, and T-wave amplitude and prolonged QT intervals; these changes were not different between adults with T1DM and healthy controls. CONCLUSIONS: Results from the current study show that adults with T1DM exhibit impaired autonomic and cardiovascular function. Additionally, novel findings highlight an effect of acute hypoglycemia to further reduce measures of autonomic and cardiovascular function similarly between adults with T1DM and healthy controls. These results suggest that acute hypoglycemia may worsen impairments in autonomic and cardiovascular control in patients with T1DM, thus increasing the risk of ventricular arrhythmias and cardiovascular mortality.
Asunto(s)
Sistema Nervioso Autónomo/fisiopatología , Diabetes Mellitus Tipo 1/fisiopatología , Hipoglucemia/fisiopatología , Adulto , Barorreflejo/fisiología , Sistema Cardiovascular/fisiopatología , Diabetes Mellitus Tipo 1/complicaciones , Femenino , Frecuencia Cardíaca/fisiología , Humanos , Hipoglucemia/etiología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Background: Stimulator of Interferon Genes (STING) is a dsDNA sensor that triggers type I inflammatory responses. Recent data from our group and others support the therapeutic efficacy of STING agonists applied intratumorally or systemically in a range of murine tumor models, with treatment benefits associated with tumor vascular normalization and improved immune cell recruitment and function within the tumor microenvironment (TME). However, such interventions are rarely curative and STING agonism coordinately upregulates expression of immunoregulatory interferon-stimulated genes (ISGs) including Arg2, Cox2, Isg15, Nos2, and Pdl1 that may limit treatment benefits. We hypothesized that combined treatment of melanoma-bearing mice with STING agonist ADU-S100 together with antagonists of regulatory ISGs would result in improved control of tumor growth vs. treatment with ADU-S100 alone. Methods: Mice bearing either B16 (BRAFWTPTENWT) or BPR20 (BRAFV600EPTEN-/-) melanomas were treated with STING agonist ADU-S100 plus various inhibitors of ARG2, COX2, NOS2, PD-L1, or ISG15. Tumor growth control and changes in the TME were evaluated for combination treatment vs ADU-S100 monotherapy by tumor area measurements and flow cytometry/transcriptional profiling, respectively. Results: In the B16 melanoma model, we noted improved antitumor efficacy only when ADU-S100 was combined with neutralizing/blocking antibodies against PD-L1 or ISG15, but not inhibitors of ARG2, COX2, or NOS2. Conversely, in the BPR20 melanoma model, improved tumor growth control vs. ADU-S100 monotherapy was only observed when combining ADU-S100 with ARG2i, COX2i, and NOS2i, but not anti-PD-L1 or anti-ISG15. Immune changes in the TME associated with improved treatment outcomes were subtle but included increases in proinflammatory innate immune cells and activated CD8+CD69+ T cells and varied between the two tumor models. Conclusions: These data suggest contextual differences in the relative contributions of individual regulatory ISGs that serve to operationally limit the anti-tumor efficacy of STING agonists which should be considered in future design of novel combination protocols for optimal treatment benefit.
Asunto(s)
Antígeno B7-H1 , Melanoma Experimental , Ratones , Animales , Proteínas Proto-Oncogénicas B-raf , Ciclooxigenasa 2 , Línea Celular Tumoral , Interferones , Microambiente TumoralRESUMEN
We previously reported that dendritic cell (DC)-based vaccines targeting antigens expressed by tumor-associated vascular endothelial cells (VECs) and pericytes effectively control tumor growth in translational mouse tumor models. In the current report, we examined whether the therapeutic benefits of such tumor blood vessel antigen (TBVA)-targeted vaccines could be improved by the cotargeting of tumor antigens in the s.c. B16 melanoma model. We also evaluated whether combination vaccines incorporating anti-PD-L1 checkpoint blockade and/or a chemokine-modulating (CKM; IFNα + TLR3-L [rintatolimod] + Celecoxib) regimen would improve T cell infiltration/functionality in tumors yielding enhanced treatment benefits. We report that DC-peptide or DC-tumor lysate vaccines coordinately targeting melanoma antigens and TBVAs were effective in slowing B16 growth in vivo and extending survival, with superior outcomes observed for DC-peptide-based vaccines. Peptide-based vaccines that selectively target either melanoma antigens or TBVAs elicited a CD8+ T cell repertoire recognizing both tumor cells and tumor-associated VECs and pericytes in vitro, consistent with a treatment-induced epitope spreading mechanism. Notably, combination vaccines including anti-PD-L1 + CKM yielded superior therapeutic effects on tumor growth and animal survival, in association with the potentiation of polyfunctional CD8+ T cell reactivity against both tumor cells and tumor-associated vascular cells and a pro-inflammatory TME.
RESUMEN
Expression of the two critical virulence factors of Vibrio cholerae, toxin-coregulated pilus and cholera toxin, is initiated at the tcpPH promoter by the regulators AphA and AphB. AphA is a winged helix DNA-binding protein that enhances the ability of AphB, a LysR-type transcriptional regulator, to activate tcpPH expression. We present here the 2.2 Å X-ray crystal structure of full-length AphB. As reported for other LysR-type proteins, AphB is a tetramer with two distinct subunit conformations. Unlike other family members, AphB must undergo a significant conformational change in order to bind to DNA. We have found five independent mutations in the putative ligand-binding pocket region that allow AphB to constitutively activate tcpPH expression at the non-permissive pH of 8.5 and in the presence of oxygen. These findings indicate that AphB is responsive to intracellular pH as well as to anaerobiosis and that residues in the ligand-binding pocket of the protein influence its ability to respond to both of these signals. We have solved the structure of one of the constitutive mutants, and observe conformational changes that would allow DNA binding. Taken together, these results describe a pathway of conformational changes allowing communication between the ligand and DNA binding regions of AphB.
Asunto(s)
Proteínas Bacterianas/química , Oxígeno/química , Transactivadores/química , Vibrio cholerae/química , Proteínas Bacterianas/genética , Clonación Molecular , Análisis Mutacional de ADN , Regulación Bacteriana de la Expresión Génica , Concentración de Iones de Hidrógeno , Mutación , Estructura Cuaternaria de Proteína , Transactivadores/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Vibrio cholerae/genética , Vibrio cholerae/patogenicidadRESUMEN
We have recently shown that intratumor (i.t.) injection of syngenic dendritic cells (DC) engineered to express the transcription factor Tbet (TBX21) promotes protective type-1 T cell-mediated immunity via a mechanism that is largely interleukin (IL)-12p70-independent. Since IL-12 is a classical promoter of type-1 immunity, the current study was undertaken to determine whether gene therapy using combined Tbet and IL-12 complementary DNA (cDNA) would yield improved antitumor efficacy based on the complementary/synergistic action of these biologic modifiers. Mice bearing established subcutaneous (s.c.) tumors injected with DC concomitantly expressing ectopic Tbet and IL12 (i.e., DC.Tbet/IL12) displayed superior (i) rates of tumor rejection and extended overall survival, (ii) cross-priming of Tc1 reactive against antigens expressed within the tumor microenvironment, and (iii) infiltration of CD8(+) T cells into treated tumors in association with elevated locoregional production of CXCR3 ligand chemokines. In established bilateral tumor models, i.t. delivery of DC.Tbet/IL12 into a single lesion led to slowed growth or regression at both tumor sites. Furthermore, DC.Tbet/IL12 pulsed with tumor antigen-derived peptides and injected as a therapy distal to the tumor site prevented tumor growth and activated robust antigen-specific Tc1 responses. These data support the translation use of combined Tbet and IL-12p70 gene therapy in the cancer setting.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Interleucina-12/genética , Neoplasias/inmunología , Neoplasias/terapia , Proteínas de Dominio T Box/genética , Animales , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Línea Celular , Quimiocinas/biosíntesis , Reacciones Cruzadas/inmunología , Células Endoteliales/inmunología , Femenino , Expresión Génica , Terapia Genética , Inmunoterapia Adoptiva , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias/genética , Neoplasias/mortalidad , Pericitos/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Hereditary spastic paraplegia (HSP) is a motor neuron disease caused by a progressive degeneration of the motor axons of the corticospinal tract. Point mutations or exon deletions in the microtubule-severing ATPase, spastin, are responsible for approximately 40% of cases of autosomal dominant HSP. Here, we report the 3.3 Å X-ray crystal structure of a hydrolysis-deficient mutant (E442Q) of the human spastin protein AAA domain. This structure is analyzed in the context of the existing Drosophila melanogaster spastin AAA domain structure and crystal structures of other closely related proteins in order to build a more unifying framework for understanding the structural features of this group of microtubule-severing ATPases.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Cristalografía por Rayos X/métodos , Animales , Drosophila melanogaster , Humanos , Microtúbulos/metabolismo , Paraplejía Espástica Hereditaria/metabolismo , EspastinaRESUMEN
New approaches consisting of 'multistage' vaccines against (TB) are emerging that combine early antigenic proteins with latency-associated antigens. In this study, HspX was tested for its potential to elicit both short- and long-term protective immune responses. HspX is a logical component in vaccine strategies targeting protective immune responses against primary infection, as well as against reactivation of latent infection, because as previously shown, it is produced during latency, and as our studies show, it elicits protection within 30 days of infection. Recent studies have shown that the current TB vaccine, bacilli Calmette-Guerin (BCG), does not induce strong interferon-γ T-cell responses to latency-associated antigens like HspX, which may be in part why BCG fails to protect against reactivation disease. We therefore tested HspX protein alone as a prophylactic vaccine and as a boost to BCG vaccination, and found that HspX purified from M. tuberculosis cell lysates protected mice against aerosol challenge and improved the protective efficacy of BCG when used as a booster vaccine. Native HspX was highly immunogenic and protective, in a dose-dependent manner, in both short- and long-term infection models. Based on these promising findings, HspX was produced as a recombinant protein in E. coli, as this would enable facile purification; however, recombinant HspX (rHspX) alone consistently failed to protect against aerosol challenge. Incubation of rHspX with mycobacterial cell lysate and re-purification following incubation restored the capacity of the protein to confer protection. These data suggest the possibility that the native form may chaperone an immunogenic and protective antigen that is mycobacteria-specific.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Chaperonas Moleculares/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Cultivadas , Escherichia coli/genética , Femenino , Humanos , Inmunidad Activa/genética , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Transgenes/genéticaRESUMEN
T-bet (Tbx21), a T-box transcription factor, has been previously identified as a master regulator of type 1 T cell polarization. We have also recently shown that the genetic engineering of human dendritic cells (DCs) to express human T-bet cDNA yields type 1-polarizing APCs in vitro (1). In the present study, murine CD11c(+) DCs were transduced with a recombinant adenovirus encoding full-length murine T-bets (DC.mTbets) and analyzed for their immunomodulatory functions in vitro and in vivo. Within the range of markers analyzed, DC.mTbets exhibited a control DC phenotype and were indistinguishable from control DCs in their ability to promote allogenic T cell proliferation in MLR in vitro. However, DC.mTbets were superior to control DCs in promoting Th1 and Tc1 responses in vitro via a mechanism requiring DC-T cell interaction or the close proximity of these two cell types and that can only partially be explained by the action of DC-elaborated IL-12p70. When injected into day 7 s.c. CMS4 sarcoma lesions growing in syngenic BALB/c mice, DC.mTbets dramatically slowed tumor progression (versus control DCs) and extended overall survival via a mechanism dependent on both CD4(+) and CD8(+) T cells and, to a lesser extent, asialoGM1(+) NK cells. DC.mTbet-based therapy also promoted superior tumor-specific Tc1 responses in the spleens and tumor-draining lymph nodes of treated animals, and within the tumor microenvironment it inhibited the accumulation of CD11b(+)Gr1(+) myeloid-derived suppressor cells and normalized CD31(+) vascular structures. These findings support the potential translational utility of DC.Tbets as a therapeutic modality in the cancer setting.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/trasplante , Ingeniería Genética/métodos , Inmunidad Innata/genética , Inyecciones Intralesiones/métodos , Sarcoma Experimental/inmunología , Sarcoma Experimental/terapia , Proteínas de Dominio T Box/administración & dosificación , Adenovirus Humanos/genética , Adenovirus Humanos/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Femenino , Regulación de la Expresión Génica/inmunología , Antígenos H-2/administración & dosificación , Antígenos H-2/genética , Humanos , Inmunofenotipificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Sarcoma Experimental/mortalidad , Sarcoma Experimental/virología , Proteínas de Dominio T Box/biosíntesis , Proteínas de Dominio T Box/genética , Transducción GenéticaRESUMEN
HLA-A2 transgenic mice bearing established HLA-A2(neg) B16 melanomas were effectively treated by intratumoral (i.t.) injection of syngeneic dendritic cells (DCs) transduced to express high levels of interleukin (IL)-12, resulting in CD8(+) T cell-dependent antitumor protection. In this model, HLA-A2-restricted CD8(+) T cells do not directly recognize tumor cells and therapeutic benefit was associated with the crosspriming of HLA-A2-restricted type-1 CD8(+) T cells reactive against antigens expressed by stromal cells [i.e., pericytes and vascular endothelial cells (VEC)]. IL-12 gene therapy-induced CD8(+) T cells directly recognized HLA-A2(+) pericytes and VEC flow-sorted from B16 tumor lesions based on interferon (IFN)-γ secretion and translocation of the lytic granule-associated molecule CD107 to the T cell surface after coculture with these target cells. In contrast, these CD8(+) T effector cells failed to recognize pericytes/VEC isolated from the kidneys of tumor-bearing HHD mice. The tumor-associated stromal antigen (TASA)-derived peptides studied are evolutionarily conserved and could be recognized by CD8(+) T cells harvested from the blood of HLA-A2(+) normal donors or melanoma patients after in vitro stimulation. These TASA and their derivative peptides may prove useful in vaccine formulations against solid cancers, as well as, in the immune monitoring of HLA-A2(+) cancer patients receiving therapeutic interventions, such as IL-12 gene therapy.