Muscle cell-mediated gene delivery to the rotator cuff

Rotator cuff lesions are one of the most common causes of upper extremity disability. Surgical therapy addresses mostly the extrinsic etiology, but not intrinsic factors such as aging, structural changes, low vascularity, and inflammatory processes. In this study, genetically engineered, highly purified muscle-derived cells (MDCs) were characterized and injected into the supraspinatus tendons of nude rats. The injected cells were monitored for 3 weeks. In vitro, the engineered, highly purified MDCs do not express vimentin; 98% of them are positive for the beta-galactosidase marker gene, and 99% hybridize with the specific pancentromeric mouse probe. beta-Galactosidase marker gene expression of the injected cells was detected up to 21 days. From day 7 after injection, the cell nuclei became spindle shaped, cells were integrated into the tendon collagen bundles, and the cells showed differentiation into vimentin-expressing fibroblastic cells. The results indicate that the rotator cuff tendon matrix and its original cellular components modulated the injected MDCs toward a fibroblastic phenotype. The compatibility and ability of MDCs to differentiate into other cell lineages, such as fibroblasts, might have high potential utility in tissue-engineering applications for tendon healing. This approach facilitates the application of muscle-derived progenitor cells and ex vivo gene therapy for the treatment of rotator cuff lesions.     

In vivo modulation of intestinal CYP3A metabolism by P-glycoprotein: studies using the rat single-pass intestinal perfusion model

P-Glycoprotein (P-gp) has been hypothesized to modulate intestinal drug metabolism by increasing the exposure of drug to intracellular CYP3A through repeated cycles of drug absorption and efflux. The rat single-pass intestinal perfusion model was used to study this interplay in vivo. N-Methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K77), a peptidomimetic cysteine protease inhibitor (CYP3A/P-gp substrate), and midazolam (CYP3A substrate) were each perfused through a segment of rat ileum alone and with the P-gp inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918). Samples were obtained continuously from the outlet perfusate and the mesenteric vein at 5-min intervals for 40 to 60 min. The parent drug and two main metabolites of K77 (N-desmethyl and N-oxide) and midazolam (1-OH and 4-OH) were quantitated by liquid chromatography/mass spectrometry. K77 appearance in the mesenteric blood (P(blood) = 5 +/- 3 x 10(-6) cm/s) was increased 3-fold with GG918, whereas midazolam permeability (P(blood) = 1.1 +/- 0.3 x 10(-4) cm/s) was unchanged by GG918. K77 metabolites were preferentially excreted into the lumen, 4-OH midazolam was found equally in lumen and blood, and 1-OH was mainly excreted into blood. The extent of metabolism was estimated by calculating the fraction metabolized = 1 - P(blood)/P(lumen) and the extraction ratio (ER) determined from the direct measurement of known metabolites as ER = sum metabolites(all)/(sum metabolites(all) + drug in blood). When P-gp was inhibited, the fraction metabolized for K77 was decreased (95 to 85%) and the ER tended toward a decrease, whereas no differences in either parameter were observed for midazolam (not a P-gp substrate). These data support a role for P-gp in modulating the extent of intestinal metabolism in vivo by controlling drug access to the enzyme.     

Enhancement of bone healing based on ex vivo gene therapy using human muscle-derived cells expressing bone morphogenetic protein 2

Molecular biological advances have allowed the use of gene therapy in a clinical setting. In addition, numerous reports have indicated the existence of inducible osteoprogenitor cells in skeletal muscle. Because of this, we hypothesized that skeletal muscle cells might be ideal vehicles for delivery of bone-inductive factors. Using ex vivo gene transfer methods, we genetically engineered freshly isolated human skeletal muscle cells with adenovirus and retrovirus to express human bone morphogenetic protein 2 (BMP-2). These cells were then implanted into nonhealing bone defects (skull defects) in severe combined immune deficiency (SCID) mice. The closure of the defect was monitored grossly and histologically. Mice that received BMP-2-producing human muscle-derived cells experienced a full closure of the defect by 4 to 8 weeks posttransplantation. Remodeling of the newly formed bone was evident histologically during the 4- to 8-week period. When analyzed by fluorescence in situ hybridization, a small fraction of the transplanted human muscle-derived cells was found within the newly formed bone, where osteocytes normally reside. These results indicate that genetically engineered human muscle-derived cells enhance bone healing primarily by delivering BMP-2, while a small fraction of the cells seems to differentiate into osteogenic cells.     

The effect of saddle design on stresses in the perineum during cycling

PURPOSE: Repetitive internal stress in the perineum has been associated with soft-tissue trauma in bicyclists. Using an engineering approach, the purpose of this study was to quantify the amount of compression exerted in the perineum for a range of saddle widths and orientations. METHODS: Computer tomography was used to create a three-dimensional voxel-based finite element model of the right side of the male perineum-pelvis. For the creation of the saddle model, a commercially available saddle was digitized and the surface manipulated to represent a variety of saddle widths and orientations. The two models were merged, and a static downward load of 189 N was applied to the model at the region representing the sacroiliac joint. For validation purposes, external stresses along the perineum-saddle interface were compared with the results of pressure sensitive film. Good agreement was found for these external stresses. The saddles were then stretched and rotated, and the magnitude and location of maximum stresses within the perineum were both recorded. In all cases, the model of the pelvis-perineum was held in an upright position. RESULTS: Stresses within the perineum were reduced when the saddle was sufficiently wide to support both ischial tuberosities. This supporting mechanism was best achieved when the saddle was at least two times wider than the bi-ischial width of the cyclist. Stresses in the anterior of the perineum were reduced when the saddle was tilted downward, whereas stresses in the posterior were reduced when the saddle was tilted upward. CONCLUSIONS: Recommendations that saddles should be sufficiently wide to support the ischial tuberosities appear to be well founded. Recommendations that saddles be tilted downward (i.e., nose down) are supported by the model, but with caution, given the limitations of the model.     

The management of subjective quality of life by short-stay hospital patients: An exploratory study

Background  

This study tested the homeostatic model of subjective quality of life in a group of 47 short stay patients as they progressed through the stages of hospitalization for surgery.     

Multitarget affinity/specificity screening of natural products: finding and characterizing high-affinity ligands from complex mixtures by using high-performance mass spectrometry

In this work we describe a high-throughput screening approach based on electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR) that rapidly interrogates the noncovalent interaction between RNA-based drug targets and components derived from a bacterial natural product library. The screening process detects molecules present in the natural product library that bind to a synthetic RNA target that mimics the prokaryotic 16S rRNA A-site, while simultaneously measuring specificity for the synthetic A-site target using a control RNA target that lacks the critical structural element of the A-site construct. This screening approach known as multitarget affinity/specificity screening (MASS) demonstrated the expected binding of paromomycin from a fractionated natural product library derived from Streptomyces rimosus sp. paromomycinus. A new molecule was observed to bind with specificity to the 16S A-site RNA construct. MS/MS characterization of this species yielded partial structural information suggesting it is an aminoglycoside consisting of a paromomycin core with one or more modified rings. This work demonstrates the tremendous utility of MASS for screening natural product fractions against macromolecular targets.     

The built environment and children's health

The built environment embraces a wide range of concepts, from the design and integrity of housing, to land-use urban planning. A high-quality environment is essential for children to achieve optimal health and development. Building and land-use policies, including the quality and design of a child's physical environment, can cause or prevent illness, disability, and injury, and can degrade or preserve natural resources. Though many common pediatric conditions such as obesity, asthma, and lead poisoning, as well as injuries, are associated with risk factors within a child's built environment, this issue has received little attention from researchers or policymakers. This new field is ripe for etiologic and prevention research, and we need pediatric advocates to speak out for children's needs within this arena.