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We describe two cases of acquired parathyroid hormone (PTH) resistance consequent to the development of serum PTH type 1 receptor (PTH1R) autoantibodies, which block PTH binding and signaling. Both cases were associated with other autoimmune manifestations, and one case was associated with atypical membranous glomerulonephritis. In vitro binding and signaling assays identified the presence of PTH1R-blocking IgG autoantibodies, which were not present in serum samples from patients with other renal or autoimmune disorders. (Funded by the Intramural Research Programs of the National Institute of Diabetes and Digestive and Kidney Diseases and others.).
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Autoanticuerpos/sangre , Hipocalcemia/etiología , Hormona Paratiroidea/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/inmunología , Adulto , Anciano , Análisis Mutacional de ADN , Femenino , Glicopéptidos/sangre , Humanos , Hipocalcemia/genética , Inmunoglobulina G/sangre , Inmunofenotipificación , Glomérulos Renales/patología , Microscopía Electrónica , Mutación , Seudohipoparatiroidismo/genéticaRESUMEN
INTRODUCTION & OBJECTIVES: To evaluate ureteral stent removal (SR) using a grasper-integrated disposable flexible cystoscope (giFC-Isiris ®, Coloplast ®) after kidney transplantation (KT), with a focus on feasibility, safety, patient experience, and costs. MATERIAL AND METHODS: All consecutive KT undergoing SR through giFC were prospectively enrolled from January 2020 to June 2023. Patient characteristics, KT and SR details, urine culture results, antimicrobial prescriptions, and the incidence of urinary tract infections (UTI) within 1 month were recorded. A micro-cost analysis was conducted, making a comparison with the costs of SR with a reusable FC and grasper. RESULTS: A total of 136 KT patients were enrolled, including both single and double KT, with 148 stents removed in total. The median indwelling time was 34 days [26, 47]. SR was successfully performed in all cases. The median preparation and procedure times were 4 min [3,5]. and 45 s[30, 60], respectively. The median Visual Analog Scale (VAS) score was 3 [1, 5], and 98.2% of patients expressed willingness to undergo the procedure again. Only one episode of UTI involving the graft (0.7%) was recorded. Overall, the estimated cost per SR procedure with Isiris ® and the reusable FC was 289.2 and 151,4, respectively. CONCLUSIONS: This prospective series evaluated the use of Isiris ® for SR in a cohort of KT patients, demonstrating feasibility and high tolerance. The UTI incidence was 0.7% within 1 month. Based on the micro-cost analysis, estimated cost per procedure favored the reusable FC.
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Cistoscopía , Remoción de Dispositivos , Equipos Desechables , Estudios de Factibilidad , Trasplante de Riñón , Stents , Humanos , Femenino , Masculino , Trasplante de Riñón/economía , Persona de Mediana Edad , Stents/economía , Remoción de Dispositivos/economía , Estudios Prospectivos , Estudios de Seguimiento , Equipos Desechables/economía , Cistoscopía/economía , Cistoscopía/métodos , Cistoscopía/instrumentación , Complicaciones Posoperatorias , Centros de Atención Terciaria , Pronóstico , Adulto , Uréter/cirugía , Infecciones Urinarias/etiología , Infecciones Urinarias/economía , Costos y Análisis de CostoRESUMEN
The majority of inherited retinal diseases (IRDs) are caused by mutations in genes expressed in photoreceptors (PRs). The ideal vector to address these conditions is one that transduces PRs in large areas of retina with the smallest volume/lowest titer possible, and efficiently transduces foveal cones, the cells responsible for acute, daylight vision that are often the only remaining area of functional retina in IRDs. The purpose of our study was to evaluate the retinal tropism and potency of a novel capsid, AAV44.9, and rationally designed derivatives thereof. We found that AAV44.9 and AAV44.9(E531D) transduced retinas of subretinally injected (SRI) mice with higher efficiency than did benchmark AAV5- and AAV8-based vectors. In macaques, highly efficient cone and rod transduction was observed following submacular and peripheral SRI. AAV44.9- and AAV44.9(E531D)-mediated GFP fluorescence extended laterally well beyond SRI bleb margins. Notably, extrafoveal injection (i.e., fovea not detached during surgery) led to transduction of up to 98% of foveal cones. AAV44.9(E531D) efficiently transduced parafoveal and perifoveal cones, whereas AAV44.9 did not. AAV44.9(E531D) was also capable of restoring retinal function to a mouse model of IRD. These novel capsids will be useful for addressing IRDs that would benefit from an expansive treatment area.
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Dependovirus/genética , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/genética , Retina/metabolismo , Transducción Genética , Animales , Dependovirus/clasificación , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Expresión Génica , Genes Reporteros , Ingeniería Genética , Vectores Genéticos/administración & dosificación , Inyecciones Intraoculares , Macaca fascicularis , Ratones , Microscopía Confocal , Oftalmoscopía , Regiones Promotoras Genéticas , Células Fotorreceptoras Retinianas Conos/metabolismo , Enfermedades de la Retina/genética , Enfermedades de la Retina/patología , Enfermedades de la Retina/terapia , Células Fotorreceptoras Retinianas Bastones/metabolismo , TransgenesRESUMEN
Genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide), cause the rare congenital disorder of glycosylation type IIb (CDG-IIb), also known as MOGS-CDG. MOGS is expressed in the endoplasmic reticulum and is involved in the trimming of N-glycans. We evaluated two siblings with CDG-IIb who presented with multiple neurologic complications and a paradoxical immunologic phenotype characterized by severe hypogammaglobulinemia but limited clinical evidence of an infectious diathesis. A shortened immunoglobulin half-life was determined to be the mechanism underlying the hypogammaglobulinemia. Impaired viral replication and cellular entry may explain a decreased susceptibility to infections.
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Agammaglobulinemia/genética , Trastornos Congénitos de Glicosilación/inmunología , Resistencia a la Enfermedad/genética , Virosis/inmunología , alfa-Glucosidasas/genética , Agammaglobulinemia/inmunología , Anticuerpos Antivirales/sangre , Niño , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/metabolismo , Femenino , Glicosilación , Humanos , Inmunoglobulinas/metabolismo , MasculinoRESUMEN
BACKGROUND: Selection of the right or left living donor kidney for transplantation is influenced by many variables. In the present multi centric study including 21 Italian transplant centres, we evaluated whether centre volume or surgical technique may influence the selection process. METHODS: Intra- and perioperative donor data, donor kidney function, and recipient and graft survival were collected among 693 mini-invasive living donor nephrectomies performed from 2002 to 2014. Centre volume (LOW, 1-50 cases; HIGH, >50 cases) and surgical technique (FULL-LAP, full laparoscopic and robotic; HA-LAP, hand-assisted laparoscopy; MINI-OPEN, mini-lumbotomy) were correlated with selection of right or left donor kidney and with donor and recipient outcome. RESULTS: HIGH-volume centres retrieved a higher rate of donor right kidneys (29.3% versus 17.6%, P < 0.01) with single artery (83.1% versus 76.4%, P < 0.05) compared with LOW-volume centres. Surgical technique correlated significantly with rate of donor right kidney and presence of multiple arteries: MINI-OPEN (53% and 13%) versus HA-LAP (29% and 22%) versus FULL-LAP (11% and 23%), P < 0.001 and P < 0.05, respectively. All donors had an uneventful outcome; donor bleeding was more frequent in LOW-volume centres (4% versus 0.9%, P < 0.05). CONCLUSIONS: Centre volume and surgical technique influenced donor kidney side selection. Donor nephrectomy in LOW-volume centres was associated with higher risk of donor bleeding.
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Selección de Donante , Hospitales de Alto Volumen/estadística & datos numéricos , Hospitales de Bajo Volumen/estadística & datos numéricos , Trasplante de Riñón/métodos , Riñón/anatomía & histología , Donadores Vivos , Nefrectomía/métodos , Recolección de Tejidos y Órganos/métodos , Femenino , Supervivencia de Injerto , Humanos , Riñón/irrigación sanguínea , Riñón/cirugía , Masculino , Persona de Mediana Edad , Factores de TiempoRESUMEN
UNLABELLED: As a genus, the dependoviruses use a diverse group of cell surface carbohydrates for attachment and entry. Despite the fact that a majority of adeno-associated viruses (AAVs) utilize sialic acid (SIA) for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, two SIA binding regions were mapped for AAV5. The first site mapped to the depression in the center of the 3-fold axis of symmetry, while the second site was located under the ßHI loop close to the 5-fold axis. Mutagenesis of amino acids 569 and 585 or 587 within the 3-fold depression resulted in elimination or alteration in SIA-dependent transduction, respectively. This change in SIA binding was confirmed using glycan microarrays. Mutagenesis of the second site identified a role in transduction that was SIA independent. Further studies of the mutants at the 3-fold site demonstrated a change in transduction activity and cell tropism in vivo as well as resistance to neutralization by a polyclonal antibody raised against the wild-type virus. IMPORTANCE: Despite the fact that a majority of AAVs utilize sialic acid for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, the sialic acid binding regions of AAV5 were identified and studied using a variety of approaches. Mutagenesis of this region resulted in elimination or alteration in sialic acid-dependent transduction in cell lines. This change in sialic acid glycan binding was confirmed using glycan arrays. Further study also demonstrated a change in transduction and activity and cell tropism in vivo as well as resistance to neutralization by antibodies raised against the wild-type virus.
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Proteínas de la Cápside/metabolismo , Dependovirus/fisiología , Ácido N-Acetilneuramínico/metabolismo , Acoplamiento Viral , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Sitios de Unión , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Línea Celular , Cristalografía por Rayos X , Análisis Mutacional de ADN , Dependovirus/química , Dependovirus/genética , Dependovirus/inmunología , Humanos , Modelos Moleculares , Mutagénesis , Transducción Genética , Tropismo ViralRESUMEN
Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvß8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy.
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Ingeniería Genética , Terapia Genética , Péptidos/genética , Anomalías Cutáneas/terapia , Proteínas de la Cápside/genética , Dependovirus/genética , Vectores Genéticos , Humanos , Integrina alfa5/genética , Queratinocitos/metabolismo , Queratinocitos/patología , Péptidos/uso terapéutico , Anomalías Cutáneas/genética , Anomalías Cutáneas/patología , Transducción Genética , TropismoRESUMEN
OBJECTIVE: Primary Sjögren's syndrome (SS) is characterized by autoimmune activation and loss of function in secretory epithelia. The present study was undertaken to investigate and characterize changes in the epithelia associated with the loss of gland function in primary SS. METHODS: To identify changes in epithelial gene expression, custom microarrays were probed with complementary RNA (cRNA) isolated from minor salivary glands (MSGs) of female patients with primary SS who had low focus scores and low salivary flow rates, and the results were compared with those obtained using cRNA from the MSGs of sex-matched healthy volunteers. The effect of bone morphogenetic protein 6 (BMP-6) on salivary gland function was tested using adeno-associated virus-mediated gene transfer to the salivary glands of C57BL/6 mice. RESULTS: A significant increase in expression of BMP-6 was observed in RNA isolated from SS patients compared with healthy volunteers. Overexpression of BMP-6 locally in the salivary or lacrimal glands of mice resulted in the loss of fluid secretion as well as changes in the connective tissue of the salivary gland. Assessment of the fluid movement in either isolated acinar cells from mice overexpressing BMP-6 or a human salivary gland cell line cultured with BMP-6 revealed a loss in volume regulation in these cells. Lymphocytic infiltration in the submandibular gland of BMP-6 vector-treated mice was increased. No significant changes in the production of proinflammatory cytokines or autoantibodies associated with SS (anti-Ro/SSA and anti-La/SSB) were found after BMP-6 overexpression. CONCLUSION: In addition to identifying BMP-6 expression in association with xerostomia and xerophthalmia in primary SS, the present results suggest that BMP-6-induced salivary and lacrimal gland dysfunction in primary SS is independent of the autoantibodies and immune activation associated with the disease.
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Proteína Morfogenética Ósea 6/metabolismo , Aparato Lagrimal/metabolismo , Glándulas Salivales/metabolismo , Síndrome de Sjögren/metabolismo , Animales , Autoanticuerpos/metabolismo , Proteína Morfogenética Ósea 6/genética , Femenino , Técnicas de Transferencia de Gen , Humanos , Aparato Lagrimal/inmunología , Aparato Lagrimal/fisiopatología , Ratones , Ratones Endogámicos C57BL , Glándulas Salivales/inmunología , Glándulas Salivales/fisiopatología , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/fisiopatología , Xerostomía/inmunología , Xerostomía/metabolismo , Xerostomía/fisiopatologíaRESUMEN
Recombinant adeno-associated viruses (AAV) have been used for therapeutic gene transfer. These vectors offer a number of advantages including resistance to the effects of pH, a broad cellular tropism, efficient gene transfer, persistence of gene expression, and little toxicity. AAV vectors; however, at high doses can induce humoral and cellular immune responses. While potentially problematic for replacement gene therapy, this effect may be advantageous for antitumor vaccination. We examined the activity of an oral and intramuscular antitumor vaccination using AAV serotypes 5 and 6 expressing a truncated neu oncogene in a neu-positive murine TUBO breast cancer model. Mice receiving a single oral administration of AAV5-neu or AAV6-neu demonstrated improved survival. Oral vaccination significantly improved survivals compared with intramuscular vaccination. Mice vaccinated with AAV6-neu survived longer than those treated with AAV5-neu. Vaccination with AAV5-neu or AAV6-neu induced both humoral and cellular immune responses against the NEU antigen. These responses were more robust in the mice undergoing oral vaccination compared with mice receiving the intramuscular vaccination. Protection from tumor was long lasting with 80% of the animals treated with oral AAV6-neu surviving a re-challenge with TUBO cells at 120 and 320 days post-vaccination. Further evaluation of AAV-based vectors as tumor vaccines is warranted.
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Neoplasias de la Mama/terapia , Vacunas contra el Cáncer/uso terapéutico , Dependovirus/genética , Vectores Genéticos , Receptor ErbB-2/uso terapéutico , Administración Oral , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Terapia Genética , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Receptor ErbB-2/genética , Transducción GenéticaRESUMEN
The growing interest in maize landraces over the past two decades has led to the need to characterize the Italian maize germplasm. In Italy, hundreds of maize landraces have been developed, but only a few of them have been genetically characterized, and even fewer are currently employed in agriculture or for breeding purposes. In the present study, 13 maize landraces of the west Emilia-Romagna region were morphologically and genetically characterized. These accessions were sampled in 1954 from three provinces, Modena, Parma, and Piacenza, during the characterization project of Italian maize landraces. The morphological characterization of these 13 accessions was performed according to the UPOV protocol CPVO/TP2/3, examining 34 phenotypic traits. A total of 820 individuals were genotyped with 10 SSR markers. The genetic characterization revealed 74 different alleles, a FST mean value of 0.13, and a Nm mean of 1.73 over all loci. Moreover, AMOVA analysis disclosed a low degree of differentiation among accessions, with only 13% of genetic variability found between populations, supporting PCoA analysis results, where the first two coordinates explained only 16% of variability. Structure analysis, supported by PCoA, showed that only four accessions were clearly distinguished for both K = 4 and 6. Italian landraces can be useful resources to be employed in maize breeding programs for the development of new varieties, adapted to different environmental conditions, in order to increase crop resilience and expand the maize cultivation area.
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INTRODUCTION: The phosphodiesterase type 5 (PDE5) inhibitors are generally well tolerated and effective for treating erectile dysfunction (ED), including in patients with significant comorbidity. Because of this benign safety profile, investigators have used PDE5 inhibitors to treat patients with ED and severe renal disease or those who have received renal transplants. AIM: To assess safety and efficacy of PDE5 inhibitors in patients receiving dialysis or renal transplants. MAIN OUTCOME MEASURES: Erectile function as assessed by the International Index of Erectile Function (IIEF) and Global Assessment Questions; adverse events (AEs). METHODS: We reviewed published studies of PDE5 inhibitors in patients receiving dialysis or renal transplants. RESULTS: In double-blind, placebo-controlled studies in patients receiving dialysis or renal transplants, sildenafil significantly improved erectile function as assessed by the IIEF, and 75-85% of patients reported improved erectile function on Global Assessment Questions; efficacy was more variable in less well-controlled studies. In >260 patients undergoing dialysis who received sildenafil in clinical studies, there were only six reported discontinuations because of AEs (headache [N=3], headache and nausea [N=1], gastrointestinal [N=1], and symptomatic blood pressure decrease [N=1]). In approximately 400 patients with renal transplants who received sildenafil, only three patients discontinued because of AEs. Vardenafil improved IIEF scores of up to 82% of renal transplant recipients in randomized, controlled studies (N=59, total), with no reported discontinuations because of AEs. Limited data also suggest benefit with tadalafil. CONCLUSIONS: ED is common in patients undergoing renal dialysis or postrenal transplant and substantially affects patient quality of life. Sildenafil and vardenafil appear to be efficacious and well tolerated in patients receiving renal dialysis or transplant.
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Disfunción Eréctil/tratamiento farmacológico , Disfunción Eréctil/fisiopatología , Fallo Renal Crónico/fisiopatología , Fallo Renal Crónico/terapia , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Adulto , Método Doble Ciego , Disfunción Eréctil/metabolismo , Humanos , Fallo Renal Crónico/metabolismo , Fallo Renal Crónico/cirugía , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Inhibidores de Fosfodiesterasa 5/farmacocinética , Ensayos Clínicos Controlados Aleatorios como Asunto , Diálisis Renal , Resultado del TratamientoRESUMEN
Propionic acidemia (PA) is rare autosomal recessive metabolic disorder caused by defects in the mitochondrially localized enzyme propionyl-coenzyme A (CoA) carboxylase. Patients with PA can suffer from lethal metabolic decompensation and cardiomyopathy despite current medical management, which has led to the pursuit of gene therapy as a new treatment option for patients. Here we assess the therapeutic efficacy of a recently described adeno-associated virus (AAV) capsid, AAV44.9, to deliver a therapeutic PCCA transgene in a new mouse model of propionyl-CoA carboxylase α (PCCA) deficiency generated by genome editing. Pcca-/- mice recapitulate the severe neonatal presentation of PA and manifest uniform neonatal lethality, absent PCCA expression, and increased 2-methylcitrate. A single injection of the AAV44.9 PCCA vector in the immediate newborn period, systemically delivered at a dose of 1e11 vector genome (vg)/pup but not 1e10 vg/pup, increased survival, reduced plasma methylcitrate, and resulted in high levels of transgene expression in the liver and heart in treated Pcca-/- mice. Our studies not only establish a versatile and accurate new mouse model of PA but further demonstrate that the AAV44.9 vectors may be suitable for treatment of many metabolic disorders where hepato-cardiac transduction following systemic delivery is desired, such as PA, and, by extension, fatty acid oxidation defects and glycogen storage disorders.
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Methylmalonic acidemia (MMA) is a severe and potentially lethal autosomal recessive inborn error of metabolism most frequently caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Proof-of-concept adeno-associated virus (AAV) gene therapy studies using mouse models of MMA have demonstrated promise for this therapeutic approach but translation to the clinic could be limited by preexisting capsid immunity and vector potency. Here we explore the efficacy of a novel clade E capsid, 44.9, as a serotype for systemic AAV gene therapy for MMA. An anti-AAV44.9 neutralizing antibody (NAb) survey in adult volunteers (n = 19) and a large cohort of MMA patients (n = 48) revealed a seroprevalence rate of â¼26% and 13%, respectively. The efficacy of AAV44.9 gene delivery was examined in two murine models of MMA, representing neonatal lethal and juvenile phenotypes of MMA. Systemic delivery of the AAV44.9-Mmut vector prevented lethality and lowered disease-related metabolites in MMA mice. Tissue biodistribution and transgene expression studies in treated MMA mice showed that AAV44.9 was efficient at transducing the liver and heart. In summary, we establish that AAV44.9 exhibits a low prevalence of preexisting NAb in humans, is highly efficacious in the treatment of clinically severe MMA mouse models and is therefore a promising vector for clinical translation.
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BACKGROUND: Aim of the present study was to verify the feasibility and accuracy of live integration of myocardial fibrosis evaluated at CCT with EAM (electro-anatomical mapping). METHODS: We prospectively enrolled a consecutive cohort of patients with clinical indication to EAM before radiofrequency catheter ablation (RFCA) of refractory ventricular tachycardia (VT) and an absolute contraindication to cardiac magnetic resonance. All patients underwent per protocol CCT for myocardial fibrosis and coronary anatomy evaluation. Diagnostic performance was assessed for myocardial fibrosis evaluation with CCT vs EAM. Live integration feasibility of CCT vs EAM was evaluated for every patients. RESULTS: A total of 19 patients were included in the present study with 323 myocardial segments analyzed for myocardial fibrosis at CCT. In all patients CCT data were successfully integrated with EAM during RFCA procedure. All patients had myocardial fibrosis correctly identified at CCT vs EAM on a per-patients basis. A diagnostic accuracy on a per-segment basis of 94.1% for detection of any type of myocardial fibrosis at CCT vs EAM was recorded. CONCLUSIONS: CCT identification of myocardial fibrosis is feasible and accurate vs EAM in a very selected high risk patients with clinical indication to RFCA of VT and contraindication to CMR.
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Cardiomiopatías , Ablación por Catéter , Taquicardia Ventricular , Ablación por Catéter/efectos adversos , Fibrosis , Humanos , Valor Predictivo de las Pruebas , Taquicardia Ventricular/diagnóstico por imagen , Taquicardia Ventricular/cirugía , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: Oncolytic viral therapy represents an alternative therapeutic strategy for the treatment of cancer. We previously described GLV-1h68, a modified Vaccinia Virus with exclusive tropism for tumor cells, and we observed a cell line-specific relationship between the ability of GLV-1h68 to replicate in vitro and its ability to colonize and eliminate tumor in vivo. METHODS: In the current study we surveyed the in vitro permissivity to GLV-1h68 replication of the NCI-60 panel of cell lines. Selected cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain. In order to identify correlates of permissity to viral infection, we measured transcriptional profiles of the cell lines prior infection. RESULTS: We observed highly heterogeneous permissivity to VACV infection amongst the cell lines. The heterogeneity of permissivity was independent of tissue with the exception of B cell derivation. Cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain and a significant correlation was found suggesting a common permissive phenotype. While no clear transcriptional pattern could be identified as predictor of permissivity to infection, some associations were observed suggesting multifactorial basis permissivity to viral infection. CONCLUSIONS: Our findings have implications for the design of oncolytic therapies for cancer and offer insights into the nature of permissivity of tumor cells to viral infection.
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Vectores Genéticos/fisiología , Virus Oncolíticos/fisiología , Virus Vaccinia/fisiología , Tropismo Viral , Línea Celular Tumoral , Análisis por Conglomerados , Medios de Cultivo , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Genes Reporteros , Células HT29 , Humanos , Transcripción Genética , Vesiculovirus/fisiología , Replicación ViralRESUMEN
Understanding the process of vector transduction has important implications for the application and optimal use of a vector system for human gene therapy. Recent studies with vectors based on adeno-associated virus type 5 (AAV-5) have shown utility of this vector system in the lung, central nervous system, muscle and eye. To understand the natural tropism of this virus and to identify proteins necessary for AAV-5 transduction, we characterized 43 cell lines as permissive or nonpermissive for AAV-5 transduction and compared the gene expression profiles derived from cDNA microarray analyses of those cell lines. A statistically significant correlation was observed between expression of the platelet-derived growth factor receptor (PDGFR-alpha-polypeptide) and AAV-5 transduction. Subsequent experiments confirmed the role of PDGFR-alpha and PDGFR-beta as receptors for AAV-5. The tropism of AAV-5 in vivo also correlated with the expression pattern of PDGFR-alpha.
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Dependovirus/genética , Dependovirus/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción Genética , Animales , Línea Celular , Receptores ErbB/genética , Receptores ErbB/metabolismo , Perfilación de la Expresión Génica , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Hipocampo/citología , Hipocampo/fisiología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Estadística como AsuntoRESUMEN
Connexin 26 (Cx26) and connexin 30 (Cx30) are encoded by two genes (GJB2 and GJB6, respectively) that are found within 50 kb in the same complex deafness locus, DFNB1. Immunocytochemistry and quantitative PCR analysis of Cx30 KO mouse cultures revealed that Cx26 is downregulated at the protein level and at the mRNA level in nonsensory cells located between outer hair cells and the stria vascularis. To explore connexin coregulation, we manipulated gene expression using the bovine adeno-associated virus. Overexpression of Cx30 in the Cx30 KO mouse by transduction with bovine adeno-associated virus restored Cx26 expression, permitted the formation of functional gap junction channels, and rescued propagating Ca(2+) signals. Ablation of Cx26 by transduction of Cx26(loxP/loxP) cultures with a Cre recombinase vector caused concurrent downregulation of Cx30 and impaired intercellular communication. The coordinated regulation of Cx26 and Cx30 expression appears to occur as a result of signaling through PLC and the NF-kappaB pathway, because activation of IP(3)-mediated Ca(2+) responses by stimulation of P2Y receptors for 20 min with 20 nM ATP increased the levels of Cx26 transcripts in Cx30 KO cultures. This effect was inhibited by expressing a stable form of the IkappaB repressor protein that prevents activation/translocation of NF-kappaB. Thus, our data reveal a Ca(2+)-dependent control in the expression of inner ear connexins implicated in hereditary deafness as well as insight into the hitherto unexplained observation that some deafness-associated DFNB1 alleles are characterized by hereditable reduction of both GJB2 and GJB6 expression.
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Conexinas/metabolismo , Oído Interno/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/fisiología , Bovinos , Conexina 26 , Conexina 30 , Conexinas/genética , Sordera/genética , Oído Interno/citología , Humanos , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer mortality for which novel gene therapy approaches relying on tumor-tropic adenoviruses are being tested. METHODS: We obtained the global transcriptional profiling of primary PDAC using RNA from eight xenografted primary PDAC, three primary PDAC bulk tissues, three chronic pancreatitis and three normal pancreatic tissues. The Affymetrix GeneChip HG-U133A was used. The results of the expression profiles were validated applying immunohistochemical and western blot analysis on a set of 34 primary PDAC and 10 established PDAC cell lines. Permissivity to viral vectors used for gene therapy, Adenovirus 5 and Adeno-Associated Viruses 5 and 6, was assessed on PDAC cell lines. RESULTS: The analysis of the expression profiles allowed the identification of two clearly distinguishable phenotypes according to the expression of interferon-stimulated genes. The two phenotypes could be readily recognized by immunohistochemical detection of the Myxovirus-resistance A protein, whose expression reflects the activation of interferon dependent pathways. The two molecular phenotypes discovered in primary carcinomas were also observed among established pancreatic adenocarcinoma cell lines, suggesting that these phenotypes are an intrinsic characteristic of cancer cells independent of their interaction with the host's microenvironment. The two pancreatic cancer phenotypes are characterized by different permissivity to viral vectors used for gene therapy, as cell lines expressing interferon stimulated genes resisted to Adenovirus 5 mediated lysis in vitro. Similar results were observed when cells were transduced with Adeno-Associated Viruses 5 and 6. CONCLUSION: Our study identified two molecular phenotypes of pancreatic cancer, characterized by a differential expression of interferon-stimulated genes and easily recognized by the expression of the Myxovirus-resistance A protein. We suggest that the detection of these two phenotypes might help the selection of patients enrolled in virally-mediated gene therapy trials.
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Adenocarcinoma , Adenoviridae , Carcinoma Ductal Pancreático , Regulación Neoplásica de la Expresión Génica , Terapia Genética/métodos , Inmunidad/fisiología , Neoplasias Pancreáticas , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma/terapia , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/terapia , Línea Celular Tumoral , Dependovirus/genética , Dependovirus/metabolismo , Perfilación de la Expresión Génica , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Interferones/inmunología , Ratones , Ratones Desnudos , Análisis por Micromatrices/métodos , Trasplante de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Selección de Paciente , Fenotipo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción Genética , Trasplante HeterólogoRESUMEN
Gene therapy vectors based on adeno-associated virus (AAV) have shown much promise in clinical trials for the treatment of a variety of diseases. However, the ability to manipulate and engineer the viral surface for enhanced efficiency is necessary to overcome such barriers as pre-existing immunity and transduction of non-target cells that currently limit AAV applications. Although single amino acid changes and peptide insertions at select sites have been explored previously, the tolerance of AAV to small deletions and tandem duplications of sequence has not been globally addressed. Here, we have generated a large, diverse library of >10(5) members containing deletions and tandem duplications throughout the viral capsid of AAV5. Four unique mutants were identified that maintain the ability to form viral particles, with one showing improved transduction on both 293T and BEAS-2B cells. This approach may find potential use for the generation of novel variants with improved and altered properties or in the identification of sites that are tolerant to insertions of targeting ligands.
Asunto(s)
Cápside/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Ingeniería Genética , Eliminación de Secuencia , Secuencias Repetidas en Tándem/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cápside/química , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Línea Celular , Biblioteca de Genes , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Transducción GenéticaRESUMEN
The loss of salivary gland function caused by radiation therapy of the head and neck or autoimmune disease such as Sjögren's syndrome is a serious condition that affects a patient's quality of life. Due to the combined exocrine and endocrine functions of the salivary gland, gene transfer to the salivary glands holds the potential for developing therapies for disorders of the salivary gland and the expression of therapeutic proteins via the exocrine pathway to the mouth, upper gastrointestinal tract, or endocrine pathway, systemically, into the blood. Recent clinical success with viral vector-mediated gene transfer for the treatment of irradiation-induced damage to the salivary glands has highlighted the need for the development of novel vectors with acinar cell tropism able to result in stable long-term transduction. Previous studies with adeno-associated virus (AAV) focused on the submandibular gland and reported mostly ductal cell transduction. In this study, we have screened AAV vectors for acinar cell tropism in the parotid gland utilizing membrane-tomato floxed membrane-GFP transgenic mice to screen CRE recombinase encoding AAV vectors of different clades to rapidly identify capsid isolates able to transduce salivary gland acinar cells. We determined that AAVRh10 and a novel isolate found as a contaminant of a laboratory stock of simian adenovirus SV15, AAV44.9, are both able to transduce parotid and sublingual acinar cells. Persistence and localization of transduction of these AAVs were tested using vectors encoding firefly luciferase, which was detected 6 months after vector administration. Most luciferase expression was localized to the salivary gland compared to that of distal organs. Transduction resulted in robust secretion of recombinant protein in both blood and saliva. Transduction was species specific, with AAVRh10 having stronger transduction activity in rats compared with AAV44.9 or AAV2 but weaker in human primary salivary gland cells. This work demonstrates efficient transduction of parotid acinar cells by AAV that resulted in secretion of recombinant protein in both serum and saliva.