RESUMEN
BACKGROUND: There is a lack of autism screening instruments for deaf or hard of hearing (DHH) adults with intellectual disability. AIMS: This study examined the diagnostic validity of the Pervasive Developmental Disorder in Mental Retardation Scale and the Diagnostic Behavioral Assessment for autism spectrum disorder - Revised in this rare population. METHODS AND PARTICIPANTS: 56 DHH adults with intellectual disability living in three specialized therapeutic communities were examined, 9 of whom met criteria for autism. OUTCOMES AND RESULTS: With minimal adaptions regarding item interpretation, both tools showed good diagnostic and high convergent validity. Items probing for difficulties in reciprocal social interaction and restricted interests were discriminant between individuals with and without autism. CONCLUSION: These data suggest that both autism screening tools are feasible and psychometrically sound when used with appropriate adaptations for DHH adults with intellectual disability.
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Trastorno del Espectro Autista , Trastorno Autístico , Discapacidad Intelectual , Adulto , Trastorno del Espectro Autista/complicaciones , Trastorno del Espectro Autista/diagnóstico , Trastorno Autístico/complicaciones , Trastorno Autístico/diagnóstico , Estudios de Factibilidad , Humanos , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/diagnóstico , Tamizaje MasivoRESUMEN
OBJECTIVES: Pentoxifylline has been used to improve sperm motility in Assisted Reproductive Technology mainly by initiating sperm motility in immotile spermatozoa samples obtained surgically. Indeed, as Intracytoplasmic Sperm Injection leads to very poor results when using immotile gametes, pentoxifylline gives better results by easing the selection of viable sperm mobilized after incubation. In 2011, the French Haute Autorité de santé decided that pentoxifylline used for in vivo purpose proposed Insufficient Medical Service and pentoxifylline was thus withdrawn from the French materia medica. We here assessed the efficacy on spermatozoa motility and the safety of papaverine, another phosphodiesterase inhibitor, for the replacement of pentoxifylline. METHODS: Sixteen frozen-thawed epididymal or testicular samples displaying no or very poor spontaneous motility (≤5% total motility) were subjected to both pentoxifylline (3.6mM) and papaverine (93µM). A duplicate Mouse Embryo Assay and an In Vitro Fertilization Mouse Assay in duplo were used to discard any toxic effect of papaverine. RESULTS: Papaverine gave better results than pentoxifylline (mean total motility: 27% vs 23%, P<0.05). No Effect Level were observed in the two different Mouse Embryo Assays performed. CONCLUSION: Papaverine is a useful tool to replace pentoxifylline in ICSI programs to select viable spermatozoa in frozen-thawed sperm samples displaying no or very poor motility.
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Epidídimo/citología , Papaverina/farmacología , Pentoxifilina , Inyecciones de Esperma Intracitoplasmáticas/métodos , Motilidad Espermática/efectos de los fármacos , Testículo/citología , Animales , Supervivencia Celular , Criopreservación , Fertilización In Vitro , Calor , Humanos , Infertilidad Masculina , Masculino , Ratones , Pentoxifilina/efectos adversos , Preservación de Semen/métodosRESUMEN
OBJECTIVE: To present the auditory implant manipulator, a navigation-controlled mechanical and electronic system which enables minimally invasive ('keyhole') transmastoid access to the tympanic cavity. MATERIALS AND METHODS: The auditory implant manipulator is a miniaturised robotic system with five axes of movement and an integrated drill. It can be mounted on the operating table. We evaluated the surgical work field provided by the system, and the work sequence involved, using an anatomical whole head specimen. RESULTS: The work field provided by the auditory implant manipulator is considerably greater than required for conventional mastoidectomy. The work sequence for a keyhole procedure included pre-operative planning, arrangement of equipment, the procedure itself and post-operative analysis. CONCLUSION: Although system improvements are necessary, our preliminary results indicate that the auditory implant manipulator has the potential to perform keyhole insertion of implantable hearing devices.
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Implantación Coclear/instrumentación , Implantes Cocleares , Robótica/instrumentación , Cirugía Asistida por Computador/instrumentación , Implantación Coclear/métodos , Diseño de Equipo , Falla de Equipo , Humanos , Apófisis Mastoides/cirugía , Procedimientos Quirúrgicos Mínimamente Invasivos/instrumentación , Procedimientos Quirúrgicos Mínimamente Invasivos/métodos , Cirugía Asistida por Computador/métodos , Tomografía Computarizada por Rayos XRESUMEN
The aim of this study was, firstly, to define the different patterns of early cleavage (EC) observed at 26 h after either IVF or intracytoplasmic sperm injection (ICSI) and, secondly, to assess the predictive value of one of these patterns, even EC (EEC), on pregnancy rate in combination with day 2 embryo score. In the first part of the study, the relationship between three different EC patterns (EEC, uneven EC and EC with fragmentation of the day 2 embryo) and embryo morphology was determined. EEC was shown to be strongly associated with good embryo morphology. In the second part of the study, it was shown that EEC used in combination with embryo score improved selection of embryos for transfer. The presence of EEC significantly (P < 0.001) enhanced mean implantation rate in all transfer categories involving identically scored embryos, in both compulsory single embryo transfers and elective single embryo transfers. Multivariate analysis demonstrated that EEC and embryo score had strong complementary predictive value for pregnancy. Based on these findings, it was concluded that even though they are associated, EEC and embryo score could be combined to increase pregnancy rate, especially in elective single embryo transfer programmes.
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Técnicas de Cultivo de Embriones , Transferencia de Embrión , Embrión de Mamíferos/citología , Fertilización In Vitro/métodos , Infertilidad/terapia , Inyecciones de Esperma Intracitoplasmáticas/métodos , Adulto , Implantación del Embrión , Femenino , Humanos , Masculino , Oocitos/citología , Embarazo , Resultado del Embarazo , Índice de EmbarazoRESUMEN
The value of early cleavage (EC) assessment is still being debated. The aim of this prospective study was to examine the predictive value of EC assessment performed exactly 26 h after insemination by IVF or intracytoplasmic sperm injection (ICSI) in a programme of elective single embryo transfer (SET) performed at day 2. If day 2 scoring demonstrated several embryos with high implantation potential, an EC embryo was transferred preferentially. EC was assessed only during normal laboratory hours so that there were two groups: EC assessed, and EC not assessed, the latter being the control. A total of 277 elective SET were performed in women under 37 years undergoing their first IVF or ICSI cycle (mean age 30.5 years, range 21-37). The overall clinical and ongoing pregnancy rates were 40.1% (111/277) and 32.9% (91/277) respectively. Significantly higher overall clinical and ongoing pregnancy rates were obtained after transfer of an EC embryo than a non-EC embryo: 49.4 versus 33.3% (P < 0.05) and 42.4 versus 25.9% (P < 0.02) respectively. However there was no significant difference between the EC assessed and control groups: 40.4 versus 39.3% and 33.2 versus 32.1 respectively. These findings confirm the value of EC assessment for selection of embryos with high implantation potential.
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Implantación del Embrión , Transferencia de Embrión , Embrión de Mamíferos/citología , Adulto , Transferencia de Embrión/normas , Desarrollo Embrionario , Femenino , Fertilización In Vitro , Humanos , Embarazo , Índice de Embarazo , Estudios ProspectivosRESUMEN
Multiple embryo transfer is associated with a high frequency of twin pregnancies with costly complications involving both mother and child. As a result high priority is currently being given to the development of single embryo transfer (SET) programs. France seems to be lagging behind Northern European countries in the development of SET and widespread use of SET will depend on convincing physicians that this policy will not have a negative impact on success rate, as has been the case for many protocols described in the literature as well as in our own experience. Our SET program includes patients less than 36 years of age undergoing their first FIV-ICSI. If two embryos showing satisfactory morphology are obtained, one is selected transferred and the other is systematically frozen. Selection for transfer is based on two criteria, i.e. observation of even early cleavage 26 hours after FIV-ICSI and evaluation of embryo morphology score on day 2. Embryo morphology score is based on the presence of four blastomeres and absence of blastomere irregularities and anucleated fragmentation. Last, a prerequisite for SET is an effective freezing program. A pregnancy rate of 13% per thawing was sufficient enough to obtain a cumulative pregnancy rate after SET (N = 205) and subsequent frozen embryo transfer (FET) similar to the cumulative pregnancy rate obtained after double embryo transfer (N = 394) and subsequent FET (46.3 vs 46.7%, NS). Twin delivery rate were respectively 2,6% after SET and 26,6% after double embryo transfer (P < 0.01).
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Criopreservación , Transferencia de Embrión , Técnicas Reproductivas Asistidas , Resultado del Tratamiento , Adulto , Transferencia de Embrión/efectos adversos , Transferencia de Embrión/tendencias , Femenino , Fertilización In Vitro , Francia , Humanos , Embarazo , Embarazo Múltiple , Técnicas Reproductivas Asistidas/estadística & datos numéricos , Técnicas Reproductivas Asistidas/tendencias , Inyecciones de Esperma Intracitoplasmáticas , GemelosRESUMEN
BACKGROUND: In age related macular degeneration and inherited dystrophies, preservation of retinal ganglion cells has been demonstrated. This finding has led to the development of various models of subretinal or epiretinal implant in order to restore vision. This study addresses the development of a polyimide subretinal electrode platform in the dystrophic P23H rat in vivo. METHODS: A technique was developed for implanting a subretinal electrode into the subretinal space and stabilising the distal extremity of the cabling on the rat cranium in order to allow future electrical stimulations of the retina. RESULTS: In vivo imaging of the retina with the scanning laser ophthalmoscope demonstrated reabsorption of the surgically induced retinal detachment and the absence of major tissue reactions. These in vivo observations were confirmed by retinal histology. The extraocular fixation system on the rat cranium was effective in stabilising the distal connector for in vivo stimulation. CONCLUSION: This study demonstrates that a retinal implant can be introduced into the subretinal space of a dystrophic rat with a stable external connection for repeatable electrical measurements and stimulation. This in vivo model should therefore allow us to evaluate the safety and efficacy of electrical stimulations on dystrophic retina.
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Terapia por Estimulación Eléctrica/instrumentación , Electrodos Implantados , Implantación de Prótesis/métodos , Degeneración Retiniana/terapia , Animales , Modelos Animales de Enfermedad , Terapia por Estimulación Eléctrica/métodos , Estudios de Factibilidad , Oftalmoscopía , Ratas , Degeneración Retiniana/patologíaRESUMEN
BACKGROUND: 360 degrees encirclement for rhegmatogenous retinal detachment (RD) was introduced over 50 years ago as a method for closing retinal breaks and relieving vitreous traction. In the absence of acknowledged guidelines, current usage varies widely. PATIENTS AND METHODS: All 360 degrees encircling bands for RDs performed between 1999 and 2004 were reviewed. Encircling procedures for phakic and recurrent RDs were analysed. RESULTS: 41 encircling bands for RD were identified from the records of 875 RD cases. Median follow-up was 6 months (range: 1 - 36). Male patients accounted for 58.5 % of the procedures. Mean age was 51.51 +/- 15.97 years. 37/41 phakic and 18/41 recurrent RDs were encircled. RD recurrence in encircled eyes occurred in 42 % of phakic eyes and in 33 % of eyes re-operated for recurrent RD. At the time of recurrence, proliferative vitreoretinopathy (PVR) was present in 41.6 % of phakic eyes, and 66 % of recurrent RD eyes. Preoperative visual acuity (VA) was 0.26 and post-operative VA 0.34. Post-operative complications due to the encirclement included pain in 4 eyes, scleral abscess in 2 eyes and scleral necrosis in 1 eye. CONCLUSION: This small retrospective review highlights the multiple indications for encircling bands including PVR, inferior breaks, multiple breaks and myopia. High overall recurrence rate (34 %) reflects the selection bias of this procedure for severe disease in our series. There is currently no consensus on the use of this technique in the management of RD.
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Procedimientos Quirúrgicos Oftalmológicos/métodos , Retina/lesiones , Retina/cirugía , Desprendimiento de Retina/cirugía , Técnicas de Sutura , Cerclaje Cervical/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Prevención Secundaria , Resultado del TratamientoRESUMEN
OBJECTIVE: Prevention of twin pregnancies using elective Single Embryo Transfer (e-SET) is now considered by many Assisted Reproductive Techniques teams as a necessity. The aim of this study was to assess the efficacy of e-SET in a prospective manner in a selective population of patients using Take Home Baby Rate per couple as principal parameter. PATIENTS AND METHODS: This prospective study was conducted from January 2003 to December 2004. Elective Single Embryo was proposed to women above 37 years in their first IVF or ICSI attempt. It was then performed only in cases when at least one embryo with high implantation potential (score-4 embryo in our embryo scoring) was obtained for transfer and one more (score-3 or score-4 embryo) was available for freezing. RESULTS: e-SET was proposed and accepted in 225 couples (25% of eligible couples and 7.8% of total population) and was possible in 96 of these). Two embryos were transferred in all other eligible patients (Double Embryo Transfer group=DET). Cumulative delivery rate after fresh embryo transfers and, if necessary, after frozen-thawed embryo transfers were 39.5% per couple e-SET group and 41.7% in DET group (NS). On the other hand, the percentage of twin pregnancies was significantly different between the two groups (2.6% vs 26.6% respectively; P<0.01). DISCUSSION AND CONCLUSION: In women younger than 37 years in their first IVF/ICSI attempt, the elective transfer of only one embryo with high implantation potential strongly allowed to avoid twin pregnancies without any significant delivery rate decrease. This transfer policy is particularly efficient in laboratories displaying good results in their embryo freezing program.
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Transferencia de Embrión , Selección de Paciente , Criopreservación , Femenino , Humanos , Embarazo , Resultado del Embarazo , Embarazo Múltiple/estadística & datos numéricos , Estudios Prospectivos , GemelosRESUMEN
3,4-Methylenedioxymethamphetamine (ecstasy), a widely used recreational drug with psychoactive properties, induces both serotonin and dopamine release in the brain. However, little is known about its intracellular effects. We previously showed that 3,4-methylenedioxymethamphetamine rewarding effects in mice were dependent upon extracellular signal-regulated kinase activation and that dorsal striatum was a critical region for mediating extracellular signal-regulated kinase-dependent Egr1 3,4-methylenedioxymethamphetamine-induced transcription. Here, we extend these findings by showing that 3,4-methylenedioxymethamphetamine is indeed able to activate extracellular signal-regulated kinase within this structure. To identify genes regulated by acute 3,4-methylenedioxymethamphetamine in the mice dorsal striatum, and selectively controlled by this kinase, we performed microarray experiments by using a selective inhibitor of extracellular signal-regulated kinase activation, SL327. Of the approximately 24,000 genes from the microarray, 27 showed altered expression after exposure to 3,4-methylenedioxymethamphetamine, and among these, 59% were partially or totally inhibited by SL327 pretreatment. Our results showed that the genes regulated by 3,4-methylenedioxymethamphetamine encode proteins that belong to transcription factors family, signaling pathways (phosphatases, cytoskeleton regulation), and synaptic functions. These early changes, and especially those controlled by extracellular signal-regulated kinase activation might play significant roles in the expression of many of the behaviors that occur following 3,4-methylenedioxymethamphetamine taking.
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Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , N-Metil-3,4-metilenodioxianfetamina/farmacología , Neostriado/efectos de los fármacos , Neostriado/enzimología , Neuronas/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Animales , Proteínas del Citoesqueleto/genética , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica/fisiología , Alucinógenos/farmacología , Masculino , Ratones , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Neuronas/enzimología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Transducción de Señal/genética , Activación Transcripcional/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
This prospective study evaluated the crude cumulative delivery rate following delayed intracytoplasmic sperm injection (ICSI) using spermatozoa recovered by testicular extraction (TESE) and intentionally frozen in men with non-obstructive azoospermia (NOA). This procedure can be termed 'cryoTESE-ICSI'. This study involved a series of 118 patients who underwent testicular biopsy for diagnosis of NOA in the period from January 1998 to December 2002. Testicular histology confirmed the diagnosis of NOA. Testicular parenchyma was obtained surgically from both testicles under general anaesthesia. Cryopreservation of spermatozoa was performed in 51 of 118 patients (43%). Ninety-nine delayed ICSI procedures were performed. Frozen-thawed suspensions were used in all cycles. Application of pentoxifylline was required to stimulate spermatozoa in 52% of cases. Fertilization, embryo transfer, and ongoing pregnancy rates were 60, 98 and 29% respectively. The crude cumulative delivery rate was 49% after two cycles and 57% after four cycles. A total of 39 healthy children were born in 29 deliveries. Thus, cryoTESE-ICSI is an effective procedure for routine use in patients with NOA. The main advantages of cryoTESE-ICSI are to (i) avoid repeated surgical biopsy, (ii) ensure the availability of spermatozoa when the ovarian stimulation cycle is begun, and (iii) allow programmed biopsy and therefore dissociate it from ICSI.
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Criopreservación/métodos , Parto Obstétrico/estadística & datos numéricos , Oligospermia/patología , Preservación de Semen/métodos , Inyecciones de Esperma Intracitoplasmáticas/métodos , Espermatozoides/fisiología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pentoxifilina/farmacología , Embarazo , Resultado del Embarazo , Índice de Embarazo , Estudios Prospectivos , Técnicas Reproductivas Asistidas , Espermatozoides/efectos de los fármacos , Testículo/citologíaRESUMEN
BACKGROUND: Since 2001, French law has permitted the use of assisted reproductive technology in human immunodeficiency virus (HIV)-1 infected women under strict conditions. This report describes a preliminary series of seropositive women who underwent assisted reproduction treatment at our facility. To minimize contamination of culture media, equipment, and therefore of male gametes and embryos, we chose to perform ICSI in all cases. The outcome of ICSI was compared with the outcome in an age-matched group of non-HIV-1-infected women. Since several previous reports have indicated that HIV infection may be associated with a decrease in spontaneous fertility, our goal was also to assess the fertility status of the HIV-1-infected women entering our ICSI programme. METHODS: The French law governing the use of assisted reproduction protocols in HIV-1-infected women was strictly applied. The inclusion criteria were absence of ongoing disease, CD4((+)) count >200 cells/mm(3), and stable HIV-1 RNA level. Since mean age at the time of ICSI was higher in HIV-1-infected women than in the overall group of non-HIV-infected women, we compared outcome data in HIV-1-infected women (group I) to a group of non-HIV-1-infected women matched with regard to age and follicle retrieval period (group II) as well as to the overall group of women who underwent ICSI at our institution (group III). RESULTS: A total of 66 ovarian stimulations was performed in 29 HIV-1-infected-infected women. The percentage of cancelled cycles was higher in infected women than in matched controls (15.2 versus 4.9%, P < 0.05). The duration of ovarian stimulation (13.3 versus 11.7 days, P < 0.05) and amount of recombinant FSH injected (2898 versus 2429 IU, P < 0.001) were also higher in infected women. The number of retrieved oocytes, mature oocytes, and embryos obtained as well as embryo quality was similar in all groups. The fertilization rate was higher in infected women than in matched controls (67 versus 60%, P < 0.01). The pregnancy rate was not significantly different between groups I and II (16.1 versus 19.6%) in spite of the fact that the number of embryos transferred was purposefully restricted in the HIV-1-infected group to minimize multiple pregnancy (2.0 versus 2.4, not significant). CONCLUSION: The results of this preliminary series of ICSI cycles in HIV-1-infected women indicate that optimal ovarian stimulation is slightly more difficult to achieve than in matched seronegative women. However, when criteria for oocyte retrieval were fulfilled, ICSI results were similar to those of age-matched controls.
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Infecciones por VIH/complicaciones , Complicaciones Infecciosas del Embarazo , Inyecciones de Esperma Intracitoplasmáticas/métodos , Adulto , Linfocitos T CD4-Positivos/metabolismo , Estudios de Casos y Controles , Femenino , VIH/metabolismo , Seropositividad para VIH , Humanos , Masculino , Oocitos/metabolismo , Inducción de la Ovulación , Embarazo , Índice de Embarazo , Embarazo de Alto Riesgo , ARN/metabolismo , ARN Viral/química , Inyecciones de Esperma Intracitoplasmáticas/legislación & jurisprudencia , Resultado del TratamientoRESUMEN
This paper describes the design of the highest affinity ligands for Grb2 SH3 domains reported so far. These compounds were designed by combining N-alkyl amino acid incorporation in a proline-rich sequence with subsequent dimerization of the peptoid sequence based on structural data and molecular modeling. Optimization of the linker size is discussed, and the N-alkyl amino acid incorporation into both monomeric halves is reported. Because the affinity for Grb2 of the optimized compounds was too high to be measured using the fluorescent modifications that they induce on the Grb2 emission spectrum, a competition assay was developed. In this test, Grb2 is pulled down from a cellular extract by the initial VPPPVPPRRR peptide bound to Sepharose beads. In the presence of competitors, the test quantifies the amount of Grb2 displaced from the beads. It has enabled us to determine a K(i) value in the 10(-10) M range for the highest affinity Grb2 peptoid analogue dimer.
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Proteínas Adaptadoras Transductoras de Señales , Peptoides/análogos & derivados , Peptoides/metabolismo , Proteínas/química , Proteínas/metabolismo , Dominios Homologos src , Alquilación , Secuencia de Aminoácidos , Animales , Línea Celular , Cricetinae , Dimerización , Proteína Adaptadora GRB2 , Humanos , Datos de Secuencia Molecular , Peptoides/síntesis química , Peptoides/química , Prolina/química , Estructura Terciaria de Proteína , Proteínas/antagonistas & inhibidores , Espectrometría de Fluorescencia , Dominios Homologos src/efectos de los fármacosRESUMEN
We have synthesised a novel cholesterol-based cationic lipid to promote DNA transfer in cells. This lipid, dimethyl hydroxyethyl aminopropane carbamoyl cholesterol iodide (DMHAPC-Chol) contains a biodegradable carbamoyl linker and a hydroxyethyl group in the polar amino head moiety and is characterised by NMR. Liposomes prepared from this lipid and dioleoyl phosphatidyl ethanolamine (DOPE) in equimolar proportion showed a weak cytotoxicity as revealed by MTT assays and are efficient to deliver plasmids DNA evaluated by the expression of reporter genes in vitro and in vivo. In this paper, we present an original method to determine the lipid concentration based on the colorimetric detection of the colipid DOPE and the measure of the molar ratio DOPE/cationic lipid in the liposome by FTIR spectroscopy. The liposomes and lipid/DNA complexes structures were characterized by transmission electron microscopy (TEM) and by quasi-elastic light scattering (QLS). TEM indicated that the complexes correspond to aggregates containing globular substructures with liposomes size. The method of immuno-gold labelling was used to detect plasmid in the complex and reveals the presence of DNA inside the aggregates. Transfection results showed efficient DNA transfer depending on the charge ratio and liposomes conditioning. Gel retardation results indicated that at a molar charge ratio between X = 1.5 and X = 2.5 (depending on the liposome conditioning), all DNA was taken by liposomes. We showed that conditioning by freeze-drying (lyophilization) facilitates storage and improves transfection efficiency. When the liposomes were lyophilized prior to DNA addition or when the complexes were subjected to freeze-thawing cycles, the obtained complexes showed a transfection with levels enhanced up to four and five-fold respectively for the lyophilized liposomes and freeze-thawed complexes. NMR was used to characterize the modifications under freezing which showed an effect on 31P spectra.
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Colesterol/administración & dosificación , ADN/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Animales , Cationes , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Colesterol/genética , Colesterol/toxicidad , ADN/genética , ADN/toxicidad , Relación Dosis-Respuesta a Droga , Femenino , Liofilización , Lípidos/administración & dosificación , Lípidos/genética , Lípidos/toxicidad , Liposomas , Ratones , Ratones Desnudos , Fosfatidiletanolaminas/administración & dosificación , Fosfatidiletanolaminas/genética , Fosfatidiletanolaminas/toxicidad , Transfección/métodosRESUMEN
This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-L-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.
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Colesterol , Técnicas de Transferencia de Gen , Liposomas , Animales , Supervivencia Celular , Colesterol/análogos & derivados , Colesterol/toxicidad , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Ratones , Ratones Desnudos , Microscopía Electrónica , Microscopía de Contraste de Fase , Trasplante de Neoplasias , Plásmidos/análisis , Polietilenglicoles , Polilisina , Ratas , Células Tumorales CultivadasRESUMEN
In vitro and in vivo transgene expression in B16-F10 melanoma cells has been investigated using an original cationic liposome prepared with triethyl aminopropane carbamoyl cholesterol iodide (TEAPC-Chol) as carrier. TEAPC-Chol/DOPE (dioleoyl phosphatidyl ethanolamine) liposomes are unilamellar, very stable and not toxic in the used concentration range. The yield in complexation with plasmid DNA can reach 100% even in the presence of fetal calf serum. The transfection level has been evaluated by luminometric measurements of luciferase expression. With TEAPC-Chol/DOPE (1:1) liposomes, a relatively high transfection level in B16-F10 cells has been observed comparing to commercial reagents. For in vivo assays, the transfection level in tumors induced in Nude mice has been optimized by studying the effects of charge ratio, of the helper lipid and of the injection volume. Results showed that TEAPC-Chol/DOPE (1:1) liposomes have improved 10-fold transfection level versus direct gene transfer of free DNA.
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Técnicas de Transferencia de Gen , Liposomas , Melanoma Experimental/genética , Animales , Supervivencia Celular/efectos de los fármacos , Colesterol , Cromatografía en Gel , ADN de Neoplasias/biosíntesis , ADN de Neoplasias/genética , Femenino , Citometría de Flujo , Genes Reporteros/genética , Proteínas Fluorescentes Verdes , Inyecciones Espinales , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Plásmidos/genética , Sales de Tetrazolio , Tiazoles , TransfecciónRESUMEN
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that need to be activated before they can function to initiate primary and secondary immune responses in vivo. DCs are also specialized to maintain peripheral tolerance to self after uptake of apoptotic material, likely corresponding to both apoptotic bodies and whole apoptotic cells. Here, we report that murine bone marrow-derived DCs can be activated in vitro by exogenous signals received from apoptotic leukemia cells expressing on the cell surface a model tumor-associated antigen. Injected in vivo, these exogenously activated DCs can function as adjuvants to protect mice against leukemia by stimulating an antigen-specific cellular-mediated cytotoxic immune response. To our knowledge, this is the first report indicating that DCs loaded with apoptotic leukemia cells protect mice against leukemia development.
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Apoptosis/fisiología , Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Leucemia L1210/inmunología , Animales , Antígenos de Neoplasias/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Antígenos CD4/genética , Antígenos CD4/inmunología , Citotoxicidad Inmunológica , Femenino , Técnicas de Transferencia de Gen , Humanos , Inmunoterapia Adoptiva , Leucemia L1210/genética , Leucemia L1210/prevención & control , Ratones , Ratones Endogámicos DBA , Fagocitosis/inmunologíaRESUMEN
We have investigated the delivery and the pathway in tumoral MCF7 cells of DNA carried by liposomes prepared from (trimethyl aminoethane carbamoyl cholesterol iodide (TMAE-Chol), a cholesterol-based cationic lipid with a quaternary ammonium on the polar head. The structure of DNA-liposome complexes depends on the length of DNA and on the lipid-DNA charge ratio X. Spherical beads constitute fine structures of the observed complexes even when they appear as aggregates. For oligonucleotide transfer, dissociation from liposomes after transfection, penetration of the oligonucleotides into nuclei, and a long resident time were observed. For plasmid transfer, a correlation between the variation in the transfection level and the ultrastructure of complexes was demonstrated. The results showed a cellular route of lipid/plasmid complexes from the beginning by endocytosis, entrapped into endosomes, released by the latter until entry in the perinuclear area, and then penetration of plasmids inside the nuclei resulting in the observed expression of the beta-galactosidase gene.
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Colesterol/metabolismo , ADN/administración & dosificación , ADN/metabolismo , Liposomas/química , Liposomas/metabolismo , Transfección/métodos , Transporte Biológico , Cationes/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , ADN/genética , ADN/ultraestructura , Endocitosis , Endosomas/metabolismo , Endosomas/ultraestructura , Fluoresceína , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Vectores Genéticos/ultraestructura , Humanos , Microscopía Confocal , Microscopía Electrónica , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Células Tumorales Cultivadas , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
AIM: To examine epiretinal membranes of proliferative diabetic retinopathy (PDR) for the presence of selective matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), in order to determine whether neovascularisation and fibrosis, characteristic of this complication of diabetes mellitus, are associated with specific anomalies of MMP or TIMP expression. METHODS: The presence of selected MMPs and TIMPs was investigated in 24 fibrovascular epiretinal membranes of PDR, and the findings compared with that observed in 21 avascular epiretinal membranes of proliferative vitreoretinopathy (PVR) and five normal retinas. Specimens were examined for deposition of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), and three tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, and TIMP-3). RESULTS: The results showed that unlike normal retina, which constitutively expresses MMP-1 and TIMP-2, a large proportion of PDR membranes (> 62%) stained for MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, and TIMP-3. There were no differences in the expression of these molecules when compared with PVR membranes. A characteristic staining for MMP-9 was observed within the perivascular matrix of PDR membranes, and there was a significant increase in TIMP-2 expression by PDR membranes (p= 0.036) when compared with PVR membranes. CONCLUSIONS: The findings that MMPs involved in degradation of fibrovascular tissue matrix, as well as TIMP-1 and TIMP-2, are found in a large proportion of PDR membranes, and that their expression does not differ from that of PVR membranes, suggest the existence of common pathways of extracellular matrix degradation in pathological processes leading to retinal neovascularisation and fibrosis.