Atresia pulmonar con septum ventricular intacto: enfoque diagnóstico y terapéutico / Pulmonary atresia with intact interventricular septum

Se describe la variabilidad, evaluación prenatal, la aproximación diagnóstica y las estrategias de tratamiento en atresia pulmonar con septum ventricular intacto, en un centro cardioquirúrgico pediátrico chileno. Una enfermedad poco común, que muestra considerable heterogeneidad morfológica, y sin reportes locales sobre estos tópicos. Estudiamos una serie consecutiva de 28 casos, en un período de cinco años (1997-2002), en el centro cardiovascular del Hospital Luis Calvo Mackenna. Las características morfológicas de cada caso fueron evaludas por revisión directa de los ecocardiogramas, angiocardiogramas, y uno de los protocolos quirúrgicos. De los 28 pacientes, la atresia fue membranosa en 82 por ciento y muscular en 18 por ciento. El ventrículo derecho fue bipartito en el 30 por ciento, unipartito en el 6 por ciento and tripartito en el 64 por ciento de los casos. Se identificaron anormalidades coronarias en el 36 por ciento, y circulación de estas dependiente del ventrículo derecho en el 7,1 por ciento. La mediana para el valor del anillo valvular tricuspideo fue de -3. El diagnóstico fue hecho en etapa fetal en seis de 28 casos (21.4 por ciento). Se tomaron las medidas para que en todos estos pacientes el parto se produjera, en un hospital cercano al centro cardiovascular. La reparación quirúrgica se realizó, basándose en predictores ecocardiográficos, el grupo con solución biventricular mostró un valor Z tricuspideo promedio de -1,1 para el grupo univentricular este valor fue de -5 (p<0,001), el índice trucúspide/mitral (T/M) fue de 0,8 y 0,4 respectivamente (p<0,001). Tres pacientes dentro del grupo biventricular requirieron, posteriormente una solución de tipo ventrículo y medio con un índice T/M medio de 0,4 (p<0,001). Existió 100 por ciento de concordancia respecto a la presencia de alteraciones coronarias, entre las técnicas ecocardiográficas y angiográfica. Este estudio muestra datos acerca de la diversidad morfológica, los esfuerzos iniciales en el diagnóstic fetal, hecho en nuestro país, además de los excelentes resultados quirúrgicos obtenidos con un enfrentamiento diagnóstico y terapéutico selectivo, en esta poco común patología

COUP-TF plays a dual role in the regulation of the ovalbumin gene.

The ovalbumin (Ov) gene contains a number of regulatory elements that control its transcriptional activity and restrict expression to avian oviduct. One major regulatory region, the steroid-dependent regulatory element (SDRE), is required for induction by estrogen and corticosterone. Another region, the negative regulatory element (NRE), downstream of the SDRE, acts primarily to repress gene expression. In addition, experiments within indicate that the binding site for the COUP transcription factor (COUP-TF) is also required for Ov gene transcription. To examine the interactions involving the SDRE, the NRE, and the COUP binding sites on Ov gene transcription, mutations in these regions were made and transfected into primary oviduct cell cultures. These experiments show that without the NRE, the SDRE is sufficient for induction by estrogen and corticosterone, irrespective of the COUP site. However, with the NRE intact, the COUP site is required for steroid induction, although without the NRE, the COUP site attenuates transcriptional activity. More interestingly, overexpression of COUP-TF1 with the Ov wild-type reporter construct alleviates the requirement for steroid hormones. These results demonstrate that the COUP site is essential and has a dual role in Ov gene transcription and that steroid hormones might directly or indirectly regulate the activity of COUP-TF1.

Repeated administration of adenoviral vectors in lungs of human CD4 transgenic mice treated with a nondepleting CD4 antibody.

The central role of CD4+ T cells in regulation of adenovirus vector-mediated immune responses has been documented previously in murine models. We analyzed the effects of a nondepleting mAb to human CD4 (CD4 mAb; Clenoliximab) on immune functions following intratracheal administration of adenoviral vectors in murine CD4-deficient mice (muCD4KO) expressing a human CD4 transgene (HuCD4 mice). Treatment of HuCD4 mice with Clenoliximab inhibited both cell-mediated and humoral immune responses to adenoviral Ags. Chronic treatment of HuCD4 mice with Clenoliximab permitted successful readministration of adenoviral vectors at least four times. The ability to readminister these vectors is associated with marked suppression of neutralizing Ab responses to viral capsid proteins. Clenoliximab also inhibited CTL and prolonged expression of the transgene. T or B cell responses to adenovirus did not emerge after the effects of a short course of Clenoliximab diminished. These data illustrate the potential utility of a nondepleting CD4 Ab in facilitating gene therapy using adenoviral vectors.

Recombinant adeno-associated virus for muscle directed gene therapy.

Although gene transfer with adeno-associated virus (AAV) vectors has typically been low, transduction can be enhanced in the presence of adenovirus gene products through the formation of double stranded, non-integrated AAV genomes. We describe the unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV). The rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers. Fluorescent in situ hybridization of muscle tissue suggests single integration sites. Neutralizing antibody against AAV capsid proteins does not prevent readministration of vector. Remarkably, no humoral or cellular immune responses are elicited to the neoantigenic transgene product E. coli beta-galactosidase. The favorable biology of rAAV in muscle-directed gene therapy described in this study expands the potential of this vector for the treatment of inherited and acquired diseases.

Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle.

Skeletal muscle is an attractive target for somatic gene transfer of both acquired and inherited disorders. Direct injection of adenoviral vectors in the skeletal muscle leads to recombinant gene expression in a large number of muscle fibers. Transgene expression has been transient in most organs and associated with substantial inflammation when experiments are performed in adult immune competent mice. In this report, we utilize a variety of in vivo and in vitro models of T and B cell function to characterize the nature of the immune response to adenoviral vectors injected into murine skeletal muscle. Cellular immunity dependent on CD4+ and CD8+ T cells contributes to the loss of recombinant gene expression and the development of localized inflammation. Antigen specific activation of T cells occurs to both viral proteins and the reporter gene beta-galactosidase. Systemic levels of neutralizing antibody to the capsid proteins of the vector are also generated. Destructive immune responses responsible for loss of transgene expression are largely directed against beta-galactosidase in that transgene expression was stable when beta-galactosidase was eliminated as a neoantigen in mice transgenic for lacZ. A strategy to prevent the cellular and humoral immunity to this therapy was developed based on transiently ablating CD4+ T cell activation at the time of vector delivery. Encouraging results were obtained when vector was administered with one of several immune modulating agents including cyclophosphamide, mAb to CD4+ cells, and mAb to CD40 ligand. These studies indicate that cellular and humoral immune responses are elicited in the context of gene therapy directed to skeletal muscle with adenoviral vectors. Transient ablation of CD4+ T cell activation prevents the effects responses of the CD8+ T and B cells.

In vivo expression of full-length human dystrophin from adenoviral vectors deleted of all viral genes.

Adenoviral vectors have been shown to effect efficient somatic gene transfer in skeletal muscle and thus offer potential for the development of therapy for Duchenne muscular dystrophy (DMD). Efficient transfer of recombinant genes has been demonstrated in skeletal muscle using recombinant adenoviruses deleted of E1. Application of this vector system to the treatment of DMD is limited by the vector immunogenicity, as well as by size constraints for insertion of recombinant genes, precluding the incorporation of a full-length dystrophin minigene construct. We describe in this study the use of helper adenovirus to generate a recombinant vector deleted of all viral open reading frames and containing a full-length dystrophin minigene. We show that this deleted vector (delta vector) is capable of efficiently transducing dystrophin in mdx mice, in myotubes in vitro and muscle fibers in vivo. Our modification of adenoviral vector technology may be useful for the development of gene therapies for DMD and other diseases.

Repression of the ovalbumin gene involves multiple negative elements including a ubiquitous transcriptional silencer.

Most eukaryotic genes are controlled by a complex array of cis-acting regulatory elements that modulate transcriptional activity. Two major regulatory elements reside in the chicken ovalbumin gene, a steroid-dependent regulatory element (SDRE, -892 to -780) and a negative regulatory element (NRE, -308 to -88). The SDRE is required for responsiveness to estrogen and glucocorticoid. The NRE appears to have the dual role of repressing transcription in the absence of steroids and of cooperating with the SDRE to activate transcription in the presence of steroids. The experiments described herein were designed to investigate the role of the NRE in repressing gene expression. Transfection of OvCAT fusion genes containing deletions in the NRE into primary oviduct cell cultures identified three elements (-308 to -256, -239 to -220, and -174 to -88) that repress transcription. Oligomers corresponding to portions of these elements also independently repress the viral thymidine kinase promoter. Interestingly, the element from -239 to -220 functions mechanistically as a silencer and shares sequence identity with silencers in other genes (TCTCTCCNA). Mobility shift studies indicated that all of the negative elements bind specific protein complexes from oviduct, none of which is appreciably affected by treatment with steroid hormones. However, oviduct-specific proteins bind to the regions from -280 to -252 and from -134 to -88, providing the first identification of potential tissue-specific elements in the ovalbumin gene. These results demonstrate that the region of DNA originally called the NRE is a multifunctional regulatory element that may be involved in several diverse regulatory activities.