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Introduction: Severe forms of short bowel syndrome (SBS) resulting in chronic intestinal failure (IF) have limited therapeutic options, all of which are associated with significant morbidities. Spring-mediated distraction enterogenesis (SMDE) uses an intraluminal self-expanding spring to generate mechanical force to induce intestinal stretching and sustained axial growth, providing a promising novel approach for patients with SBS. Previous studies have established this method to be safe and effective in small and large animal models. However, SMDE has previously not been implemented in a large, clinically relevant animal model. Methods: Juvenile mini-Yucatan pigs with 75% of their small intestine resected had intraluminal springs placed after an initial adaptive period. Morphological and histological assessments were performed on SMDE segments compared to the control region of the intestine undergoing normal adaptive responses to resection. Results: While the initial histologic adaptive response observed following resection was attenuated after a month, the SMDE segments instead augmented these adaptive changes. Specifically, intestinal length increased 2-fold in SMDE segments, and the widths of the epithelial, muscularis, and serosal layers were enhanced in SMDE compared with control segments of the same animal. This data suggests that morphologic intestinal adaptation may be enhanced with SMDE in the setting of SBS. Discussion: Here we demonstrate the successful and reproducible implementation of SMDE in a large animal model in the setting of prior intestinal resection, making SMDE a viable and novel approach for SBS to be explored further.
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Active and passive immunotherapy targeted at the amyloid-beta (Abeta) peptide has been proposed as therapeutic approach against Alzheimer's disease (AD), and efforts towards the generation and application of antibody-based reagents that are capable of preventing and clearing amyloid aggregates are currently under active investigation. Previously, we selected and characterized a new anti-Abeta1-42 phage-displayed scFv antibody, designated clone b4.4, using a non-immune human scFv antibody library and demonstrated that a peptide based on the sequence of the Ig heavy chain (VH) complementarity-determining region (HCDR3) of this antibody fragment bound to Abeta1-42)and had neuroprotective potential against Abeta1-42 mediated neurotoxicity in rat hippocampal cultured neurons. In the present study, using novel computational methods and in vitro experiments we demonstrated that b4.4 binds to the central region of Abeta1-42. We also demonstrated that this scFv antibody binds to Abeta-derived diffusible ligands (ADDLs) and neutralizes the toxicity of both fibrillar and oligomeric forms of Abeta1-42 tested in vitro in SH-SY5Y cell cultures.
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Péptidos beta-Amiloides/farmacología , Regiones Determinantes de Complementariedad/farmacología , Fragmentos de Inmunoglobulinas/farmacología , Cadenas Pesadas de Inmunoglobulina/farmacología , Fármacos Neuroprotectores/farmacología , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/inmunología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regiones Determinantes de Complementariedad/química , Mapeo Epitopo , Humanos , Fragmentos de Inmunoglobulinas/química , Cadenas Pesadas de Inmunoglobulina/química , Modelos Moleculares , Fármacos Neuroprotectores/química , Oligopéptidos/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Unión ProteicaRESUMEN
The Na(+)-glucose cotransporter (SGLT1) is expressed primarily by small intestinal epithelial cells and transports the monosaccharides glucose and galactose across the apical membrane. Here we describe the isolation and characterization of 5.3 kb of the 5'-flanking region of the SGLT1 gene by transiently transfecting reporter constructs into a variety of epithelial cell lines. A fragment (nt -235 to +22) of the promoter showed strong activity in the intestinal cell line Caco-2 but was inactive in a nonintestinal epithelial cell line (Chinese hamster ovary). Within this region, three cis-elements, a hepatocyte nuclear factor-1 (HNF-1) and two GC box sites are critical for maintaining the gene's basal level of expression. The two GC boxes bind to several members of the Sp1 family of transcription factors and, in the presence of HNF-1, synergistically upregulate transactivation of the promoter. A novel 16-bp element just downstream of one GC box was also shown to influence the interaction of Sp1 to its binding site. In summary, we report the identification and characterization of the human SGLT1 minimal promoter and the critical role that HNF-1 and Sp1-multigene members have in enhancing the basal level of its transcription in Caco-2 cells.
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Proteínas de Unión al ADN , Regulación de la Expresión Génica/fisiología , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Monosacáridos/genética , Proteínas Nucleares , Factor de Transcripción Sp1/fisiología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases/genética , Células CHO/fisiología , Células CACO-2/fisiología , Línea Celular , Cricetinae , Huella de ADN , Desoxirribonucleasa I , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Células LLC-PK1/fisiología , Datos de Secuencia Molecular , Familia de Multigenes/fisiología , Regiones Promotoras Genéticas/genética , Transportador 1 de Sodio-Glucosa , Factor de Transcripción Sp1/metabolismo , Porcinos , TransfecciónRESUMEN
The regulatory elements that control basal and activated transcriptional expression of the polymeric IgA receptor gene (pIgR) have not been defined. In this study, we performed functional analysis of the murine pIgR 5'-upstream region. Transient transfection studies identified the gene's minimal promoter to reside within 110 nucleotides upstream from the start of transcription. Substitution mutations of this region identified both a putative activator (-78 to -70) and a repressor (-66 to -52) element. DNase I footprint analysis confirmed an area of protection that spans from nucleotides -85 to -62. Mobility shift assays of the putative region confirmed binding of upstream stimulatory factor 1 (USF1) to an E box element at positions -75 and -70, representing the putative enhancer. Overexpression studies using various forms of USF suggest that both USF1 and USF2 enhance activity of the pIgR minimal promoter. We report the identification and characterization of the murine pIgR minimal promoter, as well as the critical role of USF in enhancing its basal level of transcription in Caco-2 cells.