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At least 1,441 records · Page 80Linked to original sources
PubMed2003

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.

Animals↗
PubMed1997

Ecological and evolutionary physiology of heat shock proteins and the stress response in Drosophila: complementary insights from genetic engineering and natural variation.

Classical adaptational and genetic engineering approaches offer complementary insights to understanding biological variation: the former elucidates the origins, magnitude and ecological context of natural variation, while the latter establishes which genes can underlie natural variation. Studies of the stress or heat shock response in Drosophila illustrate this point. At the cellular level, heat shock proteins (Hsps) function as molecular chaperones, minimizing aggregation of peptides in non-native conformations. To understand the adaptive significance of Hsps, we have characterized thermal stress that Drosophila experience in nature, which can be substantial. We used these findings to design ecologically relevant experiments with engineered Drosophila strains generated by unequal site-specific homologous recombination; these strains differ in hsp70 copy number but share sites of transgene integration. hsp70 copy number markedly affects Hsp70 levels in intact Drosophila, and strains with extra hsp70 copies exhibit corresponding differences in inducible thermotolerance and reactivation of a key enzyme after thermal stress. Elevated Hsp70 levels, however, are not without penalty; these levels retard growth and increase mortality. Transgenic variation in hsp70 copy number has counterparts in nature: isofemale lines from nature vary significantly in Hsp70 expression, and this variation is also correlated with both inducible thermotolerance and mortality in the absence of stress.

Animals↗
PubMed2006

Preclinical testing of tissue-engineered heart valves re-endothelialized under simulated physiological conditions.

BACKGROUND: The in vivo regeneration capacity of decellularized heart valve grafts is still controversial. The aim of this study was to evaluate function, morphological changes, and cellular composition of decellularized versus re-endothelialized ovine pulmonary valves (PV) after implantation into lambs for 1 or 3 months. METHODS AND RESULTS: PV (n=21) were decellularized using detergents. Twelve PV were repopulated with autologous jugular veins endothelial cells (ECs) in a dynamic pulsatile bioreactor under simulated physiological conditions. Morphological evaluation before implantation included histological stainings (H&E, Movat-pentachrome, von-Kossa, DAPI), immunostainings (anti-perlecan, anti-eNOS, anti-procollagen-I, anti-SM-alpha-actin), electron microscopy (EM), and DNA extraction. Decellularization led to cell-free scaffolds with preserved extracellular matrix (ECM) including basement membrane. Reseeded PV (n=5) were completely covered with ECs expressing endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). The function of orthotopically implanted decellularized and re-endothelialized PV (n=7, each) was analyzed after 1 and 3 months by echocardiography and revealed no differences in competence between both groups. A confluent EC monolayer expressing eNOS/vWF was only found in re-endothelialized PV but not in decellularized PV, whereas the valve matrices were comparable repopulated with interstitial cells expressing SM-alpha-actin and procollagen-I. More thrombotic and neointima formations were observed in decellularized PV. No signs of calcification were detected in both PV types. CONCLUSIONS: In vitro re-endothelialization of detergent-decellularized valves with autologous ECs under simulated physiological conditions significantly improves total EC valve coverage 3 months after implantation, whereas the valve repopulation with interstitial cells in vivo occurs most likely by cell migration inside the scaffold.

Animals↗
PubMed2005

Influence of stirring-induced mixing on cell proliferation and extracellular matrix deposition in meniscal cartilage constructs based on polyethylene terephthalate scaffolds.

The response of engineered meniscal cartilage constructs to stirring-induced mixing in spinner flasks was investigated. Polyethylene terephthalate scaffolds were seeded with meniscal fibrochondrocytes from 6 month-old sheep and cultured under a variety of stirring regimes for 28 days. Stirring-induced mixing increased up to 7-fold the deposition of glucosaminoglycans and up to 3-fold the deposition of collagen, when compared to static cultures. High and medium intensity stirring induced rapid cell proliferation, with maximal cell densities achieved within the first seven days of cultivation. Under these conditions, collagen and glucosaminoglycan deposition occurred predominantly in association with cell proliferation, the specific deposition rate of these biopolymers decreasing markedly after 7 days of cultivation, when the cell number reached a plateau. Constructs exposed to the highest intensity stirring had the highest levels of collagen and glucosaminoglycans and a more homogeneous cell distribution. As the success of the integration at a repair site in the knee of a meniscal construct is likely to be dependent on the cellular activity of the construct, these studies suggest that cultivation of meniscal cartilage constructs, under these conditions, should not extend for more than 7 days.

Animals↗
PubMedDate not supplied

Isolation and culture of the three vascular cell types from a small vein biopsy sample.

The availability of small-diameter blood vessels remains a significant problem in vascular reconstruction. In small-diameter blood vessels, synthetic grafts resulted in low patency; the addition of endothelial cells (EC) has clearly improved this parameter, thereby proving the important contribution of the cellular component to the functionality of any construct. Because the optimal source of cells should be autologous, the adaptation of existing methods for the isolation of all the vascular cell types present in a single and small biopsy sample, thus reducing patient's morbidity, is a first step toward future clinical applications of any newly developed tissue-engineered blood vessel. This study describes such a cell-harvesting procedure from vein biopsy samples of canine and human origin. For this purpose, we combined preexisting mechanical methods for the isolation of the three vascular cell types: EC by scraping of the endothelium using a scalpel blade, vascular smooth muscle cells (VSMC), and perivascular fibroblasts according to the explant method. Once in culture, cells rapidly grew with the high level of enrichment. The morphological, phenotypical, and functional expected criteria were maintained: EC formed cobblestone colonies, expressed the von Willebrand factor, and incorporated acetylated low-density lipoprotein (LDL); VSMC were elongated and contracted when challenged by vasoactive agents; perivascular fibroblasts formed a mechanically resistant structure. Thus, we demonstrated that an appropriate combination of preexisting harvesting methods is suitable to isolate simultaneously the vascular cell types present in a single biopsy sample. Their functional characteristics indicated that they were suitable for the cellularization of synthetic prosthesis or the reconstruction of functional multicellular autologous organs by tissue engineering.

Animals↗
PubMed2006

Dynamics of increased neutrophil adhesion to ICAM-1 after contacting immobilized IL-8.

Changing affinity of beta(2)-integrins on neutrophils for their ligands on endothelium is a critical, regulated step in the inflammatory response. In this report, the dynamics of the neutrophil response to the inflammatory chemokine interleukin-8 (IL-8) is examined. Human IL-8 was immobilized on beads and brought into contact with neutrophils selected from whole blood samples. Resulting changes in cellular adhesion were assessed by measuring the adhesion frequency between a human neutrophil and a bead coated with the endothelial ligand ICAM-1 (intercellular adhesion molecule-1). Cells engulfed the IL-8 coated beads within a few tens of seconds, and most of the cells exhibited an increase in adhesion to ICAM-1 after approximately 5 to 10 min of contact with IL-8 at room temperature (3 to 5 min at 37 degrees C). Neither monocyte chemotactic protein-1 (MCP-1) nor anti-CD45-coated beads caused any changes in adhesion to ICAM-1. IL-8 induced adhesion was blocked by antibody against CD18. At lower surface density of chemokine, approximately 20% of IL-8 coated beads adhered but were not engulfed by the cells, although the increase in adhesion for ICAM-1 was still effected. Heterogeneity in the cellular response and variability between donors was also noted.

Biomedical Engineering↗
PubMed1985

Immunopotentiating conjugates.

Current interest in the development of sub-cellular vaccines and in cancer immunotherapy has rekindled interest in immunopotentiating agents. In addition, peptide sequences of protective antigens are being synthesized or genetically engineered and it is likely that these peptides will have a small relative molecular mass and be non-immunogenic by themselves. A variety of experimental studies have shown that such protective antigens could be included in conjugated vaccines with carrier protein and/or immunopotentiator. The insertion of peptides with hydrophobic sequences into artificial lipid bilayers (liposomes) offers another mode of presentation of antigen to the immune system.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1995

Generation and characterization of a single-gene encoded single-chain immunoglobulin-interleukin-2 fusion protein.

BACKGROUND: Interleukin-2 (IL-2), a potent inducer of cellular immune responses, has been used for biological therapy of human cancer; however, the high doses of IL-2 required to mediate patients' immune responses can cause considerable systemic toxicity. The murine monoclonal antibody (MAb) CC49, which reacts with tumor-associated glycoprotein (TAG)-72, expressed on a variety of human carcinomas, has shown excellent tumor localization in recent clinical trials. OBJECTIVES: Development and characterization of a single-chain immunoglobulin-IL-2 (SCIg-IL-2) fusion protein which, by delivering IL-2 selectively to the tumor site, can serve as an effective reagent for CC49/IL-2 combination therapy. STUDY DESIGN: A single-gene encoding the SCIg-IL-2 fusion protein derived from the chimeric (c) CC49 was designed, generated and inserted in an expression vector. The monomeric single-chain protein consisted of the CC49 heavy and light chain variable domains covalently jointed through a (GGGGS)3 linker peptide. The carboxyl end of the variable domain of the light chain was linked to the amino terminus of the human gamma 1 Fc through the hinge region, and the carboxyl end of the CH3 domain was linked to the amino terminus of the human IL-2 through a GGGSGGG linker peptide. The SCIg-IL-2, expressed from the murine myeloma cells transfected with the expression construct, was characterized for its antigen-binding specificity, antibody effector functions and IL-2 biological activity. RESULTS AND CONCLUSION: Transfection of murine myeloma cells with the single-gene expression construct SCIg-IL-2 expressed a single-chain protein of approximately 70 kD, which was secreted into tissue culture fluid as a homodimer of approximately 140 kD. SCIg-IL-2 competed completely with cCC49 for binding to the TAG-72 antigen, but approximately three- to four-fold more of the SCIg-IL-2 was required to achieve levels of competition similar to those observed with the murine or chimeric CC49. With human effector cells, the fusion protein mediated lysis of TAG-72-positive human carcinoma cells. Prior treatment of human effector cells with 100 U/ml of human IL-2 enhanced the fusion protein-mediated cytolysis from 32 to 65%. At doses of > or = 1 ng/ml, the stimulatory effect of SCIg-IL-2 on IL-2 dependent murine HT-2 cell proliferation was comparable to that of the recombinant human IL-2. The single-gene construct may also facilitate inoculation of the gene in animal tissue for in vivo expression of the fusion protein.

Antibodies, Monoclonal↗
PubMed2002

Multi-layered microcapsules for cell encapsulation.

Mechanical stability, complete encapsulation, selective permeability, and suitable extra-cellular microenvironment, are the major considerations in designing microcapsules for cell encapsulation. We have developed four types of multi-layered microcapsules that allow selective optimization of these parameters. Primary hepatocytes were used as model cells to test these different microcapsule configurations. Type-1 microcapsules with an average diameter of 400 microm were formed by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 microm. Cells in these microcapsules exhibited improved cellular functions over those cultured on collagen monolayers. Type-II microcapsules were formed by encapsulating the Type-I microcapsules in another 2-5 microm ter-polymer shell and a approximately 5 microm collagen layer between the two ter-polymer shells to ensure complete cell encapsulation. Type-II microcapsules comprised of a macro-porous exoskeleton with materials such as alumina sol-gel coated on the Type-I microcapsules. Nano-indendation assay indicated an improved mechanical stability over the Type-I microcapsules. Type-IV microcapsules were created by encapsulating Type-III microcapsules in another 2-5 microm ter-polymer shell, with the aim of imparting a negatively charged smooth surface to minimize plasma protein absorption and ensure complete cell encapsulation. The permeability for nutrient exchange, cellular functions in terms of urea production and mechanical stability of the microcapsules were characterized. The advantages and limitations of these microcapsules for tissue engineering are discussed.

Animals↗
PubMed2000

Emerging cell and molecular strategies for the study and treatment of painful peripheral neuropathies.

Pharmacologic treatment for the symptoms of painful neuropathy has been problematic, because there has been limited understanding of the underlying etiologies and systemic levels that an effective dose can have on multiple side effects. The use of molecular methods, such as gene deletion from knockout mice and cellular minipumps for delivery of biologic antinociceptive molecules, has led to a better understanding of the underlying mechanisms of the induction of intractable neuropathic pain. The initiation of an excitatory cascade after injury or disease leads to the induction of various second messenger systems, loss or down-regulation of the endogenous inhibitory spinal GABA system and central sensitization, causing such pain. The development and use of cellular minipumps, immortalized cell lines bioengineered to secrete various antinociceptive molecules for the reversal of neuropathic pain, makes cellular therapy a strategy for clinical use in the next few years. The development of molecular "disimmortalization" technologies will make the use of such engineered cell lines safe for human use. Direct somatic gene transfer for neuropathic pain will eventually overcome the problems associated with transplantation of non-autologous and xenogenic cells. These virus-mediated methods, although at the early stages of evolution and use, offer large-scale production of biologic agents that can be conveniently and confidently used for the long-term relief of chronic neuropathic pain in a clinical setting, without systemic effects or surgical interventions.

Animals↗
PubMed2006

Perilipin promotes hormone-sensitive lipase-mediated adipocyte lipolysis via phosphorylation-dependent and -independent mechanisms.

Hormone-sensitive lipase (HSL) is the predominant lipase effector of catecholamine-stimulated lipolysis in adipocytes. HSL-dependent lipolysis in response to catecholamines is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin A (Peri A), an essential lipid droplet (LD)-associated protein. It is believed that perilipin phosphorylation is essential for the translocation of HSL from the cytosol to the LD, a key event in stimulated lipolysis. Using adipocytes retrovirally engineered from murine embryonic fibroblasts of perilipin null mice (Peri-/- MEF), we demonstrate by cell fractionation and confocal microscopy that up to 50% of cellular HSL is LD-associated in the basal state and that PKA-stimulated HSL translocation is fully supported by adenoviral expression of a mutant perilipin lacking all six PKA sites (Peri Adelta1-6). PKA-stimulated HSL translocation was confirmed in differentiated brown adipocytes from perilipin null mice expressing an adipose-specific Peri Adelta1-6 transgene. Thus, PKA-induced HSL translocation was independent of perilipin phosphorylation. However, Peri Adelta1-6 failed to enhance PKA-stimulated lipolysis in either MEF adipocytes or differentiated brown adipocytes. Thus, the lipolytic action(s) of HSL at the LD surface requires PKA-dependent perilipin phosphorylation. In Peri-/- MEF adipocytes, PKA activation significantly enhanced the amount of HSL that could be cross-linked to and co-immunoprecipitated with ectopic Peri A. Notably, this enhanced cross-linking was blunted in Peri-/- MEF adipocytes expressing Peri Adelta1-6. This suggests that PKA-dependent perilipin phosphorylation facilitates (either direct or indirect) perilipin interaction with LD-associated HSL. These results redefine and expand our understanding of how perilipin regulates HSL-mediated lipolysis in adipocytes.

Adipocytes↗
PubMed2005

Characterization of transcript levels for matrix molecules and proteases in ruptured human anterior cruciate ligaments.

An improved understanding of cellular responses during normal anterior cruciate ligament (ACL) function or repair is essential for clinical assessments, understanding ligament biology, and the implementation of tissue engineering strategies. The present study utilized quantitative real-time RT-PCR combined with univariate and multivariate statistical analyses to establish a quantitative database of marker transcript expression that can provide a "blueprint" of ACL wound healing. Selected markers (collagen types I and III, biglycan, decorin, MMP-1, MMP-2, MMP-9, and TIMP-1) were assessed from 33 torn ACLs harvested during reconstructive surgery. Trends were observed between postinjury period and marker expressions. Significant correlations between marker expression existed and were most prominent between collagen types I and III. Canonical correlation analysis established a relationship between patient demographics and a combination of all marker expressions. The currently observed trends and correlations may assist in identifying appropriate tissue samples and provide a baseline information of marker expression level that can support in vitro optimization of environmental cues for ligament tissue engineering application.

Adolescent↗
PubMedDate not supplied

Perfusion of medium with supplemented growth factors changes metabolic activities and cell morphology of hepatocyte-nonparenchymal cell coculture.

To develop a feasible perfusion-type bioartificial liver device, perfusion of hepatocyte-nonparenchymal cell (NPC) cocultures with medium supplemented with hepatocyte growth factor (HGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) was carried out. On day 1 of culture, perfusion at a constant shear stress of 1.3 dyn/cm2 enhanced ammonia metabolic and urea synthetic activities of hepatocytes. These enhanced activities were sustained up to day 7 only when growth factors were present. In contrast, no beneficial effects of growth factors on these activities were observed in static cultures. In perfusion cultures, three-dimensional cell aggregates were formed. On the surface of these aggregates, flattened cell layers composed mainly of NPCs were found, and the central cluster of cell aggregates was composed of round-shaped hepatocytes and reticulin fibrils. These observations strongly suggested that the reconstruction of different types of liver cells and connective tissues formed tissue-mimicking cell aggregates in the perfusion culture that was able to modulate the liver-specific functions of hepatocytes. Thus, perfusion culture conditions of the hepatocyte--NPC coculture system should be appropriately designed to induce suitable reconstruction of the cultured cells for use as a bioartificial liver device.

Albumins↗
PubMed2006

Malignant melanoma: genetics and therapeutics in the genomic era.

Cell for cell, probably no human cancer is as aggressive as melanoma. It is among a handful of cancers whose dimensions are reported in millimeters. Tumor thickness approaching 4 mm presents a high risk of metastasis, and a diagnosis of metastatic melanoma carries with it an abysmal median survival of 6-9 mo. What features of this malignancy account for such aggressive behavior? Is it the migratory history of its cell of origin or the programmed adaptation of its differentiated progeny to environmental stress, particularly ultraviolet radiation? While the answers to these questions are far from complete, major strides have been made in our understanding of the cellular, molecular, and genetic underpinnings of melanoma. More importantly, these discoveries carry profound implications for the development of therapies focused directly at the molecular engines driving melanoma, suggesting that we may have reached the brink of an unprecedented opportunity to translate basic science into clinical advances. In this review, we attempt to summarize our current understanding of the genetics and biology of this disease, drawing from expanding genomic information and lessons from development and genetically engineered mouse models. In addition, we look forward toward how these new insights will impact on therapeutic options for metastatic melanoma in the near future.

Animals↗
PubMed2000

Contractility affects stress fiber remodeling and reorientation of endothelial cells subjected to cyclic mechanical stretching.

We studied the effect of contractility on stress fiber remodeling and orientation response of human aortic endothelial cells subjected to cyclic mechanical stretching. The cells were grown on silicone membranes subjected to 10% cyclic pure uniaxial stretching in the presence or absence of 2,3 butanedione monoxime (BDM), a proven inhibitor of cellular contractility. It was found that treatment of the cells with BDM (40 mM) abolished stress fibers and blocked cell reorientation in response to cyclic stretching, indicating that contractility is required for these two cellular responses. When cells were stretched in the presence of N-acetylcysteine (NAG, 20 mM), a hydrogen peroxide (H2O2) scavenger, stress fibers were still formed and the cells reoriented--but more slowly. Specifically, compared with untreated cells, NAG treated cells after 0.5, 1, and 3 h of 10% stretching had significantly (p<0.005) less skewed orientation distributions than those of untreated cells. After the cells were treated with both NAG (20 mM) and nordihydroguaiaretic acid (NDGA, 50 microM), another antioxidant, however, stress fibers were abolished and cell reorientation was completely blocked. These results indicate that reactive oxygen species (ROS), including H2O2, affect stress fiber remodeling and reorientation of endothelial cells in response to cyclic stretching. We suggest that the effect of ROS on stress fiber remodeling and cell reorientation is due to the ability of ROS to regulate cellular contractility, which is crucial for these cellular responses.

Acetylcysteine↗