Biomedical subjects

D A Lee

Publications and source records attributed to D A Lee.

At least 19 recordsLinked to original sources

PubMed2004

Increased presence of cells with multiple elongated processes in osteoarthritic femoral head cartilage.

OBJECTIVE: This study examined the morphology of chondrocytes in articular cartilage from osteoarthritic (OA) and non-OA human femoral heads and in particular the appearance of a sub-population of cells with multiple elongated processes radiating up to 30 microm into the extracellular matrix. METHODS: Cartilage explants were removed from 8 anatomical sites over the surface of OA (n=6) and non-OA (n=5) femoral heads. Cells were labeled for vimentin intermediate filaments and visualized using epi-fluorescence and confocal microscopy. The percentage of cells with elongated processes was correlated with macroscopic and histological indicators of osteoarthritis. RESULTS: Cells with processes accounted for less than 10% of the total cell population in non-OA cartilage. By contrast, in the peripheral regions of the OA femoral head these cells accounted for 20-45% of the total cell population, the differences being statistically significant. These peripheral areas are habitually non-load bearing and were also the most likely to show gross fibrillation and pannus formation. A statistically significant correlation was demonstrated between the percentage of cells with processes and the histological extent of the OA degradation, quantified in terms of the Mankin score. CONCLUSIONS: The extension of cell processes, which may be associated with localized breakdown of the pericellular matrix, will undoubtedly alter numerous aspects of cell function including phenotypic expression and mechanotransduction. Hence these significant changes in chondrocyte morphology are likely to have important implications for the aetiology of osteoarthritis and the development of potential treatment strategies.

Cartilage, Articular↗

PubMed2003

Dynamic compression counteracts IL-1 beta-induced release of nitric oxide and PGE2 by superficial zone chondrocytes cultured in agarose constructs.

OBJECTIVE: To examine the effect of IL-1 beta-induced *NO and PGE(2)release by stimulated superficial and deep chondrocyte/agarose constructs subjected to mechanical compression. DESIGN: Chondrocyte sub-populations were seeded separately in agarose constructs and cultured unstrained, within a 24-well tissue culture plate, for 48 h in medium supplemented with IL-1 beta and/or L-N-(1-iminoethyl)-ornithine (L-NIO). In a separate experiment, superficial and deep cell containing constructs were subjected to 15% dynamic compressive strain at 1 Hz, for 48 h, in the presence or absence of IL-1 beta and/or L-NIO. Nitrite was measured using the Griess assay, PGE(2)release was determined using an EIA kit and [3H]-thymidine and 35SO(4)incorporation were assessed by TCA and alcian blue precipitation, respectively. RESULTS: The current data reveal that IL-1 beta significantly enhanced *NO and PGE(2)release for superficial chondrocytes, an effect reversed with L-NIO. *NO and PGE(2)levels did not significantly change by deep cells in the presence of IL-1 beta and/or L-NIO. For both cell sub-populations, IL-1 beta inhibited cell proliferation whereas proteoglycan synthesis was not affected. Dynamic compression inhibited the release of *NO and PGE(2)in the presence and absence of IL-1 beta, for cells from both sub-populations. L-NIO reduced *NO and enhanced PGE(2)release for superficial zone chondrocytes, an effect not observed for deep cells in response to dynamic compression. The magnitude of stimulation of [3H]-thymidine incorporation was similar for both cell sub-populations and was not influenced by L-NIO, indicating an z.rad;NO-independent pathway. The dynamic compression-induced stimulation of 35SO(4)incorporation was enhanced with L-NIO for IL-1 beta-stimulated deep cells, indicating an *NO-dependent pathway. CONCLUSION: The present findings suggest that dynamic compression inhibits *NO and PGE(2)release in IL-1 beta-stimulated superficial cells via distinct pathways, a significant finding that may contribute to the development of intervention strategies for the treatment of inflammatory joint disorders.

Animals↗

PubMed2003

Dynamic compressive strain inhibits nitric oxide synthesis by equine chondrocytes isolated from different areas of the cartilage surface.

REASONS FOR PERFORMING STUDY: Chondrocytes within articular cartilage respond to the mechanical stresses associated with normal joint loading via a series of signalling pathways. Specific biomolecules, such as nitric oxide (NO), have been implicated in these mechanotransduction processes. It has been shown that the synthesis of NO can be inhibited by dynamic compressive strain of chondrocytes in vitro which, in turn, leads to an up-regulation of specific metabolic parameters. HYPOTHESIS: Chondrocytes isolated from different joint locations and seeded in agarose constructs respond in a distinct manner to the application of dynamic compression. METHODS: Chondrocytes were isolated separately from the equine patella groove and the femoral condyle, representing high loaded areas (HLA) and low loaded areas (LLA), respectively, of 6 specimens of different ages. The cells were seeded in agarose constructs and cultured either in an unstrained state or strained under dynamic loading at 1 Hz for 48 h. The synthesis of nitric oxide (NO), proteoglycan synthesis and chondrocyte proliferation were assessed. RESULTS: Equine chondrocytes were found to synthesise significant basal levels of NO, regardless of topographical origin or age of tissue. Marked differences in both proteoglycan synthesis and cell proliferation were, however, revealed between the 2 chondrocyte subpopulations. Dynamic compression inhibited NO synthesis but significant alterations in proteoglycan synthesis and cell proliferation were apparent in a minority of cases. CONCLUSIONS AND POTENTIAL RELEVANCE: The differential response of the subpopulations of chondrocytes derived from the HLA and LLA provides a potential mechanism which enables the biomechanical demands of differing joint regions to be maintained.

Agar↗

PubMed2003

Dermal fibroblasts respond to mechanical conditioning in a strain profile dependent manner.

Fibroblasts within tissues are exposed to a dynamic mechanical environment, which influences the structural integrity of both healthy and healing soft tissues. Various systems have been proposed to subject such cells to mechanical stimulation in culture. However the diverse nature of the studies, in terms of the strain profiles and the cell types, makes direct comparisons almost impossible. The present study addresses this issue by examining the metabolic response of two cell types subjected to three well defined strain profiles.A young fibroblast cell population, represented by HuFFs, showed both greater cell proliferation and collagen production than adult dermal fibroblasts under unstrained conditions. The three strain profiles produced differing effects on both cell types. Uniaxial strains enhanced [(3)H]-thymidine incorporation for both cell types, whilst biaxial strains either inhibited or had no effect on its incorporation. In contrast, [(3)H]-proline incorporation was inhibited under biaxial and uniaxial strains for the adult fibroblasts, whilst the HuFF cells showed a small increase in proline incorporation under non-uniform and uniaxial strains.

Adult↗

PubMed2003

Development of a technique to determine strains in tendons using the cell nuclei.

Tenocytes detect mechanical stimuli in vivo, and respond through mechanotransduction pathways to initiate matrix remodelling in tendons. Due to the crimped nature of tendon fascicles, the strain field throughout is non-homogeneous. The present study has developed a means to quantify the local strain fields within a fascicle by monitoring the relative movement and deformation of fluorescently labelled tenocyte nuclei. A stage mounted test rig was designed to apply tensile strain to fascicles. Rat tail and bovine extensor tendons were harvested for analysis, and the cell nuclei stained and visualised using an inverted confocal microscope. As the fascicles were subjected to gross strains of up to 5%, the movement of selected tenocyte nuclei were recorded. Results from a series of cell nuclei from both tendon sources revealed that local strains were significantly less than the applied strain. The nuclei length to width ratio, an indicator of cell deformation, also increased with applied strain, most significantly between 2 and 3% applied strain.

Animals↗

PubMed2002

Cell and nucleus deformation in compressed chondrocyte-alginate constructs: temporal changes and calculation of cell modulus.

Mechanical loading is essential for the homeostasis of articular cartilage and may be necessary for achieving functional tissue engineered cartilage repair using isolated cells seeded in scaffolds such as alginate. Chondrocyte mechanotransduction is poorly understood, but may involve cell deformation and associated distortion of intracellular organelles. The present study used confocal microscopy to examine cell and nucleus morphology in isolated chondrocytes compressed in alginate constructs. Compression of 2% alginate resulted in cell deformation from a spherical to an oblate ellipsoid morphology with conservation of cell volume. Cell deformation was associated with deformation, to a lesser degree, of the nucleus. Despite constant cell deformation over a 25 min period of static compression, the nucleus deformation reduced significantly, particularly in the axis perpendicular to the applied compression. Constructs made of a lower alginate concentration exhibited a reduced compressive modulus with an altered cellular response to compression. In 1.2% alginate, compression resulted in cell deformation which was initially of a similar magnitude to that in 2% alginate but subsequently reduced over a 60 min period reflecting the viscoelastic behaviour of the gel. This phenomenon enabled the calculation of a stress-strain relationship for the cell with an estimated Young's modulus value of approx. 3 kPa.

Alginates↗

PubMed2002

Deformation properties of articular chondrocytes: a critique of three separate techniques.

This paper presents a series of techniques, which examine the deformation characteristics of bovine articular chondrocytes. The direct contact approach employs well established methodology, involving AFM and micropipette aspiration, to yield structural properties of local regions of isolated chondrocytes. The former technique yields a non-linear response with increased structural stiffness in a central location on a projected image of the chondrocyte. A simple viscoelastic model can be used with data from the micropipette aspiration technique to yield a mean value of Young's modulus, which is similar to that recently reported (Jones et al., 1999). An indirect approach is also described, involving the response of chondrocytes seeded within compressed agarose constructs. For 1% agarose constructs, the resulting cell strain, yields a gross cell modulus of 2.7 kPa. The study highlights the difficulties in establishing unique mechanical parameters, which reflect the deformation behaviour of articular chondrocytes.

Animals↗

PubMed2001

Dynamic compression inhibits the synthesis of nitric oxide and PGE(2) by IL-1beta-stimulated chondrocytes cultured in agarose constructs.

Both mechanical loading and interleukin-1beta (IL-1beta) are known to regulate metabolic processes in articular cartilage through pathways mediated by nitric oxide ((*)NO) and PGE(2). This study uses a well-characterized model system involving isolated chondrocytes cultured in agarose constructs to test the hypothesis that dynamic compression alters the synthesis of (*)NO and PGE(2) by IL-1beta-stimulated articular chondrocytes. The data presented demonstrate for the first time that dynamic compression counteracts the effects of IL-1beta on articular chondrocytes by suppressing both (*)NO and PGE(2) synthesis. Inhibitor experiments indicated that the dynamic compression-induced inhibition of PGE(2) synthesis and stimulation of proteoglycan synthesis were (*)NO mediated, while compression-induced stimulation of cell proliferation was (*)NO independent. The inhibition of (*)NO and PGE(2) by dynamic compression is a finding of major significance that could contribute to the development of novel strategies for the treatment of cartilage-degenerative disorders.

Animals↗

PubMed2001

Effects of tissue transglutaminase on retinoic acid-induced cellular differentiation and protection against apoptosis.

Retinoic acid (RA) and its various synthetic analogs affect mammalian cell growth, differentiation, and apoptosis. Whereas treatment of the human leukemia cell line HL60 with RA results in cellular differentiation, addition of the synthetic retinoid, N-(4-hydroxyphenyl) retinamide (HPR), induces HL60 cells to undergo apoptosis. Moreover, pretreatment of HL60 cells as well as other cell lines (i.e. NIH3T3 cells) with RA blocks HPR-induced cell death. In attempting to discover the underlying biochemical activities that might account for these cellular effects, we found that monodansylcadaverine (MDC), which binds to the enzyme (transamidase) active site of tissue transglutaminase (TGase), eliminated RA protection against cell death and in fact caused RA to become an apoptotic factor, suggesting that the ability of RA to protect against apoptosis is linked to the expression of active TGase. Furthermore, it was determined that expression of exogenous TGase in cells exhibited enhanced GTP binding and transamidation activities and mimicked the survival advantage imparted by RA. We tested whether the ability of this dual function enzyme to limit HPR-mediated apoptosis was a result of the ability of TGase to bind GTP and/or catalyze transamidation and found that GTP binding was sufficient for the protective effect. Moreover, excessive transamidation activity did not appear to be detrimental to cell viability. These findings, taken together with observations that the TGase is frequently up-regulated by environmental stresses, suggest that TGase may function to ensure cell survival under conditions of differentiation and cell stress.

3T3 Cells↗

PubMed2001

Chondrocyte deformation within mechanically and enzymatically extracted chondrons compressed in agarose.

Within articular cartilage, the chondron microenvironment will influence chondrocyte behaviour and response to loading. Chondrons were extracted from intact cartilage using either mechanical homogenisation (MC) or enzymatic digestion (EC) and cell and matrix morphology in unstrained and compressed agarose constructs was examined. Isolated chondrocytes (IC) were used for comparison. Immunolocalisation of type VI collagen and keratan sulphate revealed differences in the structure of the pericellular microenvironment such that MC most closely resembled chondrons in situ. The unstrained cell diameters of IC and EC were larger than MC at day 1 and increased significantly over a 7 day culture period. In contrast, cell diameters for MC remained constant. Compression of constructs at day 1 resulted in cell deformation for IC and EC but not MC. The two chondron extraction methods yielded chondrons of differing matrix morphology and associated differences in cell size and cellular response to load. The results indicate that the pericellular microenvironment of MC initially possessed a greater mechanical integrity than that of EC. Although these differences may be reduced with time in culture, characterisation of mechanically isolated chondrons suggests that the stiffness of the chondrons in situ may be greater than previous estimates.

Animals↗

PubMed2001

Temporal changes in cytoskeletal organisation within isolated chondrocytes quantified using a novel image analysis technique.

This paper examines temporal changes in the organisation of the cytoskeleton within isolated articular chondrocytes cultured for up to 7 days in agarose constructs. Fluorescent labelling and confocal microscopy were employed to visualise microtubules (MT), vimentin intermediate filaments (VIF) and actin microfilaments (AMF). To quantify the degree of cytoskeletal organisation within populations of cells, a novel image analysis technique has been developed and fully characterised. Organisation was quantified in terms of an Edge Index, which reflects the density of 'edges' present within the confocal images as defined by a Sobel digital filter. This parameter was shown to be independent of image intensity and, for all three cytoskeletal components, was validated statistically against a visual assessment of organisation. Both MT and VIF exhibited fibrous networks extending throughout the cytoplasm, while AMF appeared as punctate units associated with the cell membrane. The use of the Edge Index parameter revealed statistical significant temporal variation, in particular associated with VIF and AMF. These findings indicate the possibility of cytoskeletal mediated temporal variation in many aspects of cell behaviour following isolation from the intact tissue. Furthermore, the image analysis techniques are likely to be useful for future studies aiming to quantify changes in cytoskeletal organisation.

Animals↗

PubMed2001

Cyclophotocoagulation: a report by the American Academy of Ophthalmology.

OBJECTIVE: This document describes cyclophotocoagulation procedures for glaucoma and examines the evidence to answer key questions about patient selection, and efficacy of transscleral and endoscopic techniques. METHODS: A literature search conducted for the years 1968 to 2000 retrieved 130 citations. The author reviewed 34 of these articles and selected 19 for the panel methodologist to review and rate according to the strength of evidence. A Level I rating is assigned to properly conducted, well-designed, randomized clinical trials; a Level II rating is assigned to well-designed cohort and case-control studies; and a Level III rating is assigned to case series and poorly designed prospective and retrospective studies, including case-control studies. RESULTS: The predominant problem with all studies on cyclophotocoagulation is the lack of a uniform definition of success, which makes comparisons difficult. One randomized controlled trial (Level I evidence) compared the efficacy of transscleral cyclophotocoagulation with noncontact Nd:YAG and semiconductor diode laser. It found no significant difference between the two, although a significant problem was the variability allowed with laser parameters. Most of the literature consists of noncomparative case series that provide evidence that is limited and often not convincing. CONCLUSION: Cyclophotocoagulation is indicated for patients with refractory glaucoma who have failed trabeculectomy or tube shunt procedures, patients with minimal useful vision and elevated intraocular pressure, and patients who have no visual potential and need pain relief (based on Level III evidence). It may be useful for patients whose general medical condition precludes invasive surgery or who refuse more aggressive surgery (i.e., filter or tube). It is also useful in emergent situations, such as the acute onset of neovascular glaucoma. There is insufficient evidence to definitively compare the relative efficacy of the cyclophotocoagulation procedures for glaucoma. It is the panel's opinion, however, that semiconductor diode systems appear to possess the best combination of effectiveness (based on Level III evidence), portability, expense, and ease of use at this time.

Academies and Institutes↗

PubMed2001

Effectiveness and safety of brimonidine as adjunctive therapy for patients with elevated intraocular pressure in a large, open-label community trial.

PURPOSE: To determine the effectiveness and safety of brimonidine when used in combination with one or more glaucoma medications in patients with glaucoma or ocular hypertension. METHODS: Post hoc analysis of a multicenter, 2-month, open-label, prospective, clinical trial in which 2,335 patients received brimonidine as monotherapy, replacement therapy, or combination therapy. Patients who received brimonidine as combination therapy (n = 554) were examined in the current analysis. Efficacy was determined by the reduction in intraocular pressure (IOP) from baseline before the addition of brimonidine. Safety and tolerability parameters included adverse events and quality-of-life measures (e.g., ocular comfort, energy level, breathing ability, night vision). RESULTS: Addition of brimonidine to preexisting regimens resulted in an overall mean additional reduction in IOP of 17.9% (4.26 mm Hg) at month 2 (P < 0.001). Significant additional IOP lowering was provided by brimonidine in combination with each preexisting regimen evaluated (P < or = 0.061). When brimonidine was added to monotherapy with a nonselective beta-blocker, the mean additional IOP reduction was 15.5% (3.61 mm Hg, P < 0.001). Addition of brimonidine to latanoprost monotherapy provided a 32.2% (5.89 mm Hg) mean additional IOP reduction (P < 0.001). Addition of brimonidine to combination regimens that included latanoprost provided additional mean decreases in LOP ranging from 15.5% (3.63 mm Hg, P < 0.002) to 20.1% (6.62 mm Hg, P < 0.001). All quality-of-life parameters remained high or improved during the study. Mild to moderate adverse events were reported in 5.23% of patients (29 of 552); 98.1% of physicians rated brimonidine adjunctive therapy as good or excellent. CONCLUSIONS: Brimonidine, when added to existing glaucoma regimens, safely and effectively lowered IOP in this community-based trial.

Adrenergic alpha-Agonists↗

PubMed2001

Compressive deformation and damage of muscle cell subpopulations in a model system.

To study the effects of compressive straining on muscle cell deformation and damage an in vitro model system was developed. Myoblasts were seeded in agarose constructs and cultured in growth medium for 4 days. Subsequently, the cells were allowed to fuse into multinucleated myotubes for 8 days in differentiation medium, resulting in a population of spherical myoblasts (50%), spherical myotubes (35%), and elongated myotubes (15%) with an overall viability of 90%. To evaluate cell deformation upon construct compression half-core shaped constructs were compressed up to 40% strain and the resulting cell shape was assessed from confocal scans through the central plane of spherical cells. The ratio of cell diameters measured parallel and perpendicular to the axis of compression was used as an index of deformation (DI). The average DI of myoblasts decreased with strain level (0.99+/-0.03, 0.70+/-0.04, and 0.56+/-0.10 at 0%, 20%, and 40% strain), whereas for myotubes DI decreased up to 20% strain and then remained fairly constant (0.99+/-0.06, 0.55+/-0.06, 0.50+/-0.11). The discrepancy in DI between spherical myoblasts and myotubes at 20% strain was explained by the relative sensitivity of the cell membrane to buckling, which is more pronounced in the myotubes. Sustained compression up to 24 h at 20% strain resulted in a significant increase in cell damage with time as compared to unstrained controls. Despite differences in membrane buckling no difference in damage between myoblasts and spherical myotubes was observed over time, whereas the elongated myotubes were more susceptible to damage.

Animals↗

PubMed2001

Mechanical compression influences intracellular Ca2+ signaling in chondrocytes seeded in agarose constructs.

Ca2+ signaling forms part of a possible mechanotransduction pathway by which chondrocytes may alter their metabolism in response to mechanical loading. In this study, a well-characterized model system utilizing bovine articular chondrocytes embedded in 4% agarose constructs was used to investigate the effect of physiological mechanical compressive strain applied after 1 and 3 days in culture. The intracellular Ca2+ concentration was measured by use of the ratiometric Ca2+ indicator indo 1-AM and confocal microscopy. A positive Ca2+ response was defined as a percent increase in Ca2+ ratio above a preset threshold. A significantly greater percentage of cells exhibited a positive Ca2+ response in strained constructs compared with unstrained controls at both time points. In strained constructs, treatment with either Ga3+ or EGTA significantly reduced the number of positive Ca2+ responders compared with untreated controls. These results represent an important step in understanding the physiological role of intracellular Ca2+ in chondrocytes under mechanical compression.

Algorithms↗

PubMed2001

The role of shame and guilt in traumatic events: a clinical model of shame-based and guilt-based PTSD.

Post-traumatic stress disorder is currently classified as an anxiety disorder with fear as the predominant emotion. This has led to the development of treatment techniques such as exposure aimed at alleviating fear. This article highlights the need to address other emotional responses, in particular shame and guilt, when assessing and treating PTSD. Hence, it presents two clinical models of shame-based PTSD and guilt-based PTSD. These models are offered as aids to clinicians in assessing and formulating cases of PTSD where shame and guilt are salient issues. The models highlight the importance of assessing meaning in the context of pre-existing schemas and address two pathways to the development of shame and/or guilt: schema congruence and schema incongruence. Several treatment implications are drawn from the models.

Guilt↗

PubMed2001

Eye conditions among 5- to 7-year-old Asian-Pacific Islander schoolchildren in Southern California.

BACKGROUND: The prevalence rates and specific risk factors for development of eye conditions among Asian-Pacific Islander schoolchildren are unknown. This information is necessary to understand their eye care needs and to improve access to eye care for this rapidly growing population. METHODS: Data obtained from a computer database of vision screening examinations conducted by the UCLA Mobile Eye Clinic from October 1987 through December 1996 were analyzed for 2,687 Asian-Pacific Islander schoolchildren between 5 and 7 years of age. RESULTS: Myopia (8.9%) and astigmatism (15.8%) represented the majority of visual disorders. Mean myopic refractive error was -1.21 +/- 0.83 D among bilateral myopes. Color-vision deficiency was prevalent among 2.8% of male children, extraocular muscle imbalance among 3.0% of children, and amblyopia among 1.0% of children. All other conditions were present in less than 4% of children screened. CONCLUSIONS: Asian-Pacific Islander schoolchildren may be at higher risk for development of juvenile myopia than white, Hispanic, and African-American children. Prevalence rates of other disorders are comparable to those obtained for other ethnic groups. This information can be used in planning to meet the eye care needs of Asian-Pacific Islander schoolchildren.

Asia↗

PubMed2000

The effect of integrin antibodies on the attachment and proliferation of human Tenon's capsule fibroblasts.

The integrins are protein heterodimers consisting of noncovalently associated alpha and beta subunits. The adhesive interactions mediated by integrins are necessary for cellular survival and proliferation. In this study we investigated the effects of three different integrin antibodies on the proliferation of human Tenon's capsule fibroblasts in tissue culture. Human Tenon's capsule fibroblasts were cultured into 96 well plates and treated with different concentrations (ranging from 10(-6) to 1 microg ml(-1)) of three different integrin antibodies: human integrin alpha-2 antibody, human integrin alpha-3 antibody and human integrin alpha-5/FnR (fibronectin receptor) antibody. Coulter counter, hexosaminidase, and 3H-thymidine assays were used to determine the inhibitory effects of these integrin antibodies on ocular fibroblasts on days 0 (attachment), 1,3 and 7 following antibody treatment. The concentration of each antibody required to produce a proliferation 50% less than the control (ID50) was calculated for each assay. With respect to attachment, all three antibodies studied displayed some inhibitory activity. All three antibodies also displayed dose-dependent antiproliferative properties, especially at the highest concentration tested after 7 days of exposure. The integrin alpha-2 antibody was the most potent of the inhibitors, followed by the integrin alpha-3 antibody, with the integrin alpha-5 antibody being the least potent antibody tested. In addition, the anti-proliferative activities of the integrin alpha-2 and integrin alpha-3 antibodies increased with increasing incubation time. In conclusion, these integrin antibodies demonstrated some inhibitory effects on the attachment and proliferation of human Tenon's capsule fibroblasts in culture. Further investigation will be required to determine whether integrin antibodies can significantly limit scar formation in vivo without significant toxicity.

Antibodies↗