Author Correction: Computer keyboard interaction as an indicator of early Parkinson's disease.

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Computer keyboard interaction as an indicator of early Parkinson's disease.

Parkinson's disease (PD) is a slowly progressing neurodegenerative disease with early manifestation of motor signs. Objective measurements of motor signs are of vital importance for diagnosing, monitoring and developing disease modifying therapies, particularly for the early stages of the disease when putative neuroprotective treatments could stop neurodegeneration. Current medical practice has limited tools to routinely monitor PD motor signs with enough frequency and without undue burden for patients and the healthcare system. In this paper, we present data indicating that the routine interaction with computer keyboards can be used to detect motor signs in the early stages of PD. We explore a solution that measures the key hold times (the time required to press and release a key) during the normal use of a computer without any change in hardware and converts it to a PD motor index. This is achieved by the automatic discovery of patterns in the time series of key hold times using an ensemble regression algorithm. This new approach discriminated early PD groups from controls with an AUC = 0.81 (n = 42/43; mean age = 59.0/60.1; women = 43%/60%;PD/controls). The performance was comparable or better than two other quantitative motor performance tests used clinically: alternating finger tapping (AUC = 0.75) and single key tapping (AUC = 0.61).

Dual proline labeling protocol for individual "baseline" and "response" biosynthesis measurements in human articular cartilage.

OBJECTIVE: The heterogeneity of biosynthesis in human-derived cartilage explants poses a challenge to its use in experiments. The aim of this study was to determine the consistency with which two consecutive measures of biosynthesis could be made in individual human articular cartilage explants using a dual proline radiolabeling protocol. METHODS: Full-thickness cartilage explants were harvested from young bovine or human (total knee replacement) tibial plateaus. Two consecutive measurements of biosynthesis were obtained by measuring (3)H-proline and (14)C-proline incorporation. Each sample's ratio of (14)C-/(3)H-proline incorporation was computed. For comparison to traditional experimental designs, the (14)C-proline incorporation ratio was computed for adjacent cartilage samples. The number of samples needed to observe a change in the proline incorporation ratio of 10, 20, and 50% was determined for both methods. RESULTS: The dual-label ratio was consistent across samples from the same plateau [95% confidence interval (CI): +/-20% (human) and +/-30% (bovine) of median]. Adjacent human sample pairs had much greater variability in their (14)C-proline incorporation (95% CI: +/-50% of median). Adjacent bovine sample pairs had CIs that were similar in magnitude to those for the dual-label approach. In the human plateaus, ratio changes of 10, 20 and 50% could be detected using dramatically fewer samples than the adjacent pair method. For bovine samples, the two methods required a similar number of samples per group. CONCLUSION: The consistency of the dual-label approach may overcome the difficulties in studying the effects of interventions on biosynthesis in human cartilage in vitro.

Long-term culture of tissue engineered cartilage in a perfused chamber with mechanical stimulation.

One approach to functional tissue engineering of cartilage is to utilize bioreactors to provide environmental conditions that stimulate chondrogenesis in cells cultured on biomaterial scaffolds. We report the combined use of a three-dimensional in vitro model and a novel bioreactor with perfusion of culture medium and mechanical stimulation in long-term studies of cartilage development and function. To engineer cartilage, scaffolds made of a non-woven mesh of polyglycolic acid (PGA) were seeded with bovine calf articular chondrocytes, cultured for an initial 30-day period under free swelling conditions, and cultured for an additional 37 day period in one of the three groups: (1) free-swelling, (2) static compression (on 24 h/day, strain control, static offset 10%), and (3) dynamic compression (on 1 h/day; off 23 h/day; strain control, static offset 2%, dynamic strain amplitude 5%; frequency 0.3 Hz). Constructs were sampled at timed intervals and assessed with respect to structure, biochemical composition, and mechanical function. Mechanical simulation had little effect on the compositions, morphologies and on mechanical properties of construct interiors discs, but it resulted in distincly different properties of the peripheral rings and face sides. Contructs cultured with mechanical loading maintained their cylindrical shape with flat and parallel top and bottom surfaces, and retained larger amounts of GAG. The modular bioreactor system with medium perfusion and mechanical loading can be utilized to define the conditions of cultivation for functional tissue engineering of cartilage.

Magnetic resonance imaging of relative glycosaminoglycan distribution in patients with autologous chondrocyte transplants.

RATIONALE AND OBJECTIVES: Autologous chondrocyte transplantation (ACT) is a potential treatment for full-thickness chondral lesions in the knee. Delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) has recently been developed as a sensitive and specific measure of cartilage glycosaminoglycans (GAGs). Under the conditions of dGEMRIC, T1 is directly related to the GAG concentration. Our aim for this study was to demonstrate the potential of dGEMRIC to evaluate ACT implants. METHODS: Eleven ACT implants were studied 2 to 24 months postoperatively by dGEMRIC. T1 values from three regions of interest were obtained to examine GAG content (1) in the implant, (2) in native cartilage adjacent to the implant, and (3) in native cartilage further removed from the implant (as "control"). RESULTS: One implant failed and therefore was not included. Four of the implants were studied between 2 and 6 months postoperatively and showed low T1 (GAG), less than 80% of the control native cartilage. Five of the six implants studied between 12 and 24 months postoperativley showed T1 (GAG) comparable to (>80%) of control. One 18-month graft showed low T1 comparable to the surrounding native cartilage, with normal GAG seen in cartilage far from the graft site. The GAG index (T1 values of the graft normalized to control) from the group of implants 6 months or less was 59% +/- 5% of control, whereas those at 12 to 24 months were 91% +/- 18% of control. The two groups were statistically different with a P value of 0.005. CONCLUSIONS: The GAG level in grafts that were implanted for less than 12 months appeared to be lower than that in the remote cartilage. At 12 months or greater, the grafts in this study had GAG levels that were comparable to both the adjacent and remote cartilage. This preliminary study of ACT implants has shown that it is feasible to apply the dGEMRIC technique in patients with ACT as a way to obtain information related to the composition of grafts. These results provide motivation and the pilot data with which to design further clinical studies.

Biochemical (and functional) imaging of articular cartilage.

Over the coming decades nondestructive biochemical imaging by magnetic resonance imaging (MRI) will provide an adjunct or surrogate for the destructive histologic and biochemical assays used today. A number of MRI methods demonstrate image contrast that, although influenced by the biochemical composition, is not normally specific to a particular measure of the biochemical state. The most widely used of these is T2-weighted imaging, which variably reveals collagen ultrastructure, hydration (or collagen content), and, to a lesser extent, glycosaminoglycan (GAG) concentration (each of these biochemical metrics is an important determinant of the functional integrity of cartilage). The lack of specificity of this technique (and others discussed herein) confounds efforts to improve strategies for evaluating cartilage. However, three methods permit a very specific measure of the cartilage biochemical state. Each of these three methods, explored in detail in this article, is rooted in a biophysical theory that relates the image signal intensity to a specific biochemical feature. Proton-density imaging directly measures water content (hydration), a parameter that might increase approximately 5% with significant degeneration. Magic-angle imaging, in which the angle dependence of T2 is measured, can provide a specific measure of collagen (or macromolecular) ultrastructure. The difficulty in getting the angle dependence presently precludes its use clinically. Delayed gadolinium-enhanced MRI of cartilage provides a specific measure of the distribution of GAGs. This method measures the distribution of a charged contrast agent, which in turn reflects the distribution of charge associated with GAG. This technique can be used in a clinical setting, and ongoing studies will explore its utility in monitoring therapeutic efficacy and disease progression. Although none of these techniques are presently in routine clinical use, emerging data provide promise that the future will see patient-specific biochemical analysis of cartilage, an outcome almost unimaginable 20 years ago.

Holding forces of single-particle dielectrophoretic traps.

We present experimental results and modeling on the efficacy of dielectrophoresis-based single-particle traps. Dielectrophoretic forces, caused by the interaction of nonuniform electric fields with objects, have been used to make planar quadrupole traps that can trap single beads. A simple experimental protocol was then used to measure how well the traps could hold beads against destabilizing fluid flows. These were compared with predictions from modeling and found to be in close agreement, allowing the determination of sub-piconewton forces. This not only validates our ability to model dielectrophoretic forces in these traps but also gives insight into the physical behavior of particles in dielectrophoresis-based traps. Anomalous frequency effects, not explainable by dielectrophoretic forces alone, were also encountered and attributed to electrohydrodynamic flows. Such knowledge can now be used to design traps for cell-based applications.

Protocol issues for delayed Gd(DTPA)(2-)-enhanced MRI (dGEMRIC) for clinical evaluation of articular cartilage.

Biochemical and histologic data have validated the technique of delayed gadolinium-enhanced MRI, in which the T(1) values of cartilage after penetration of Gd(DTPA)2-allow assessment of the glycosaminoglycan (GAG) component of articular cartilage. This work describes the factors that have been found to be important for the practical implementation of the technique: 1) Exercise immediately after intravenous contrast administration was necessary for effective penetration of the contrast into the articular cartilage; 2) double-dose contrast was better than single-dose; 3) after contrast administration, a time window of 30-90 min for the hip, and 2-3 hr for all compartments of the knee proved to be appropriate for assessing articular cartilage; and 4) in some cases of hypointensities in the subchondral patellar bone, decreased penetration of the contrast agent into cartilage from bone was found. With the protocol described, ROIs on T(1) images were reproducible within 15% on two separate imaging sessions, and initial clinical studies demonstrated the possible applications of the technique. Magn Reson Med 45:36-41, 2001.

MRI techniques in early stages of cartilage disease.

Cartilage degenerative diseases affect millions of people. Our understanding of these diseases and our ability to establish efficacious treatment strategies have been confounded by the difficulty of nondestructively evaluating the state of cartilage. Imaging strategies that allow visualization of cartilage integrity would revolutionize the field by allowing us to visualize early stages of degeneration and thus to evaluate predisposing factors for cartilage disease and changes resulting from interventions (eg, therapies) in culture studies, tissue-engineered systems, animal models, and in vivo in humans. Here we briefly review current state-of-the-art MRI strategies relevant to understanding and following treatment in early cartilage degeneration. We review MRI as applied to the assessment of the whole joint, of cartilage as a whole (as an organ), of cartilage tissue, and of cartilage molecular composition and structure. Each of these levels is amenable to assessment by MRI and offers different information that, in the long run, will serve as an important element of cartilage imaging.

Alveolar cell stretching in the presence of fibrous particles induces interleukin-8 responses.

Inhalation of fibrous particulates is strongly associated with lung injury, but the molecular and cellular mechanisms that could explain the fiber-induced pathogenesis are not fully understood. We hypothesized that the physical stress exerted on the alveolar epithelium by the deposited fibers is greatly enhanced by the tidal cyclic motion of the epithelial cells that is associated with breathing, and that this initial mechanical interaction triggers a subsequent cell response. To test this hypothesis, we developed a dynamic model of fiber-induced cell injury using a cell-stretcher device. We exposed a cyclically stretched monolayer of the human alveolar epithelial cell line A549 to glass or crocidolite asbestos fibers for 8 h and then measured the production of the proinflammatory cytokine interleukin (IL)-8 as a readout of fiber-induced cell injury. Cyclic stretching significantly increased IL-8 production in the fiber-treated cultures, suggesting that the physical stress on the cells caused by the fibers was indeed enhanced by the motion. Coating of the asbestos fibers with fibronectin, a glycoprotein abundant in the alveolar lining fluid, further increased the fiber-induced cell response when the cells were cyclically stretched. This response was, however, significantly reduced by introducing into the culture medium, before fiber treatment, soluble RGD (Arg-Gly-Asp)-containing peptides, which specifically block binding to integrin receptors upon RGD attachment. These results suggested that adhesive interactions between protein-coated fibers and cell surface molecules are involved in the fiber-induced pathogenic process. Our novel findings indicate the importance of physical insults in fiber-induced cell stress, and bring to the forefront the need to study the mechanisms involved in this process.

Monitoring glycosaminoglycan replenishment in cartilage explants with gadolinium-enhanced magnetic resonance imaging.

We previously devised a magnetic resonance imaging method that allows for the nondestructive and quantitative determination of glycosaminoglycan concentration in excised cartilage. The technique measures the concentration of the charged contrast agent Gd-DTPA2- (gadolinium diethylenetriamine-pentaacetic acid) equilibrated within cartilage, from which the tissue distribution of glycosaminoglycan can be calculated. The goals of our study were to determine the practicality of nondestructively monitoring glycosaminoglycan concentration in cartilage explants over a long-term culture period and to determine if glycosaminoglycan could be restored to glycosaminoglycan-depleted cartilage explants maintained in long-term culture. To meet our objectives, we harvested bovine cartilage explants, treated them initially with trypsin to reduce the glycosaminoglycan concentration, and cultured them for as long as 8 weeks. Images depicting glycosaminoglycan concentration were calculated from magnetic resonance images acquired at selected intervals during the trypsinization process and the subsequent culture period. The results indicate that gadolinium-enhanced magnetic resonance imaging can follow the reduction of glycosaminoglycan concentration over the course of enzymatic digestion and the replenishment of glycosaminoglycan over several weeks of culture and that cultured cartilage explants are capable of restoring glycosaminoglycan to 85% of its initial concentration. Of particular interest, samples cultured for 5 weeks indicated a depth dependence of glycosaminoglycan regeneration to values similar to the initial physiologic distribution. Thus, this magnetic resonance imaging method may be a very powerful means for exploring the spatial and temporal evolution of glycosaminoglycan in cartilage.

Nondestructive imaging of human cartilage glycosaminoglycan concentration by MRI.

Despite the compelling need mandated by the prevalence and morbidity of degenerative cartilage diseases, it is extremely difficult to study disease progression and therapeutic efficacy, either in vitro or in vivo (clinically). This is partly because no techniques have been available for nondestructively visualizing the distribution of functionally important macromolecules in living cartilage. Here we describe and validate a technique to image the glycosaminoglycan concentration ([GAG]) of human cartilage nondestructively by magnetic resonance imaging (MRI). The technique is based on the premise that the negatively charged contrast agent gadolinium diethylene triamine pentaacetic acid (Gd(DTPA)2-) will distribute in cartilage in inverse relation to the negatively charged GAG concentration. Nuclear magnetic resonance spectroscopy studies of cartilage explants demonstrated that there was an approximately linear relationship between T1 (in the presence of Gd(DTPA)2-) and [GAG] over a large range of [GAG]. Furthermore, there was a strong agreement between the [GAG] calculated from [Gd(DTPA)2-] and the actual [GAG] determined from the validated methods of calculations from [Na+] and the biochemical DMMB assay. Spatial distributions of GAG were easily observed in T1-weighted and T1-calculated MRI studies of intact human joints, with good histological correlation. Furthermore, in vivo clinical images of T1 in the presence of Gd(DTPA)2- (i.e., GAG distribution) correlated well with the validated ex vivo results after total knee replacement surgery, showing that it is feasible to monitor GAG distribution in vivo. This approach gives us the opportunity to image directly the concentration of GAG, a major and critically important macromolecule in human cartilage.

Microfabrication in biology and medicine.

Microfabrication uses integrated-circuit manufacturing technology supplemented by its own processes to create objects with dimensions in the range of micrometers to millimeters. These objects can have miniature moving parts, stationary structures, or both. Microfabrication has been used for many applications in biology and medicine. These applications fall into four domains: tools for molecular biology and biochemistry, tools for cell biology, medical devices, and biosensors. Microfabricated device structures may provide significantly enhanced function with respect to a conventional device. Sometimes microfabrication can enable devices with novel capabilities. These enhancing and enabling qualities are conferred when microfabrication is used appropriately to address the right types of problems. Herein, we describe microfabrication technology and its application to biology and medicine. We detail several classes of advantages conferred by microfabrication and how these advantages have been used to date.

Quantitative measurement of the biological response of cartilage to mechanical deformation.

Cartilage functionality is defined, in part, in terms of the ability of the extracellular matrix to support a mechanical load. It has been shown that such mechanical loading can influence the biological response of the chondrocytes that are embedded in the extracellular matrix. Cultured tissue explants have served as useful models for studying such chondrocyte-mediated responses to mechanical deformation. The explant paradigm facilitates the control of mechanical and biological variables that may influence cellular behavior. This chapter presents the means for assessing the biologic response of cartilage to controlled mechanical stimuli, and lays the foundation to further explore the response of cartilage to mechanical stimuli in the presence of other factors, such as cytokines and growth factors (e.g., IL-1ß, TGF-ß, IGF).

Osteoblast cytoskeletal modulation in response to mechanical strain in vitro.

The structural integrity of microfilaments has been shown to be necessary for the signal transduction of mechanical stimuli within osteoblasts. Qualitative and quantitative changes within the cytoskeleton of osteoblasts may therefore be crucial components of the signal transduction processes of these cells in response to mechanical stimulation. Avian osteoblasts were strained with a device that deforms a flexible, cell-laden membrane at a defined frequency and intensity in a uniform biaxial manner. We examined the effects of mechanical strain on the accumulation of protein and the expression of the major cytoskeletal elements and specific integrin-binding (arginine-glycine-aspartic acid) proteins of these cells. Mechanical strain increased the level of total extracellular matrix-accumulated fibronectin by approximately 150% and decreased that of osteopontin by approximately 60% but had no quantifiable effect on the accumulation of beta1 integrin subunit or collagen type I. An examination of the major elements of the cytoskeleton demonstrated that neither the level of actin nor that of the intermediate filament protein vimentin changed; however, the amount of tubulin decreased by approximately 75% and the amount of vinculin, a major protein of focal adhesion complexes, increased by approximately 250%. An analysis of protein synthesis by two-dimensional gel electrophoresis of [35S]methionine-labeled cytoskeletal proteins demonstrated that the changes in the accumulation of vinculin and tubulin resulted from their altered synthesis. Messenger RNA analysis confirmed that the changes in accumulation and protein synthesis observed for vinculin, fibronectin, and osteopontin were controlled at a pretranslational level. Immunofluorescent microscopy demonstrated that mechanical strain led to increased formation and thickening of actin stress fibers, with a commensurate dissociation in microtubules and a clear increase in levels of vinculin at the peripheral edges of the cells. In conclusion, the elevated rate of synthesis and the increased accumulation of vinculin and fibronectin, as well as the increase in the number and size of stress fibers and focal adhesion complexes, suggest that mechanical strain leads to a coordinated change both in the cytoskeleton and in extracellular matrix proteins that will facilitate tighter adhesion of an osteoblast to its extracellular matrix.

The incidence of a positive ice water test in bladder outlet obstructed patients: evidence for bladder neural plasticity.

PURPOSE: The ice water test triggers a C fiber, capsaicin sensitive spinal micturition reflex. We postulated that the ice water test is positive in a high proportion of patients with compared to those without bladder outlet obstruction. MATERIALS AND METHODS: Prospective evaluation of 111 consecutive patients was undertaken. Symptoms of urgency, urge incontinence, nocturia and daytime frequency as well as the presence of neurological disease were obtained from history and physical examination. Fluorourodynamics, including ice water cystometry, and pressure-flow studies were done for all 111 subjects. Obstruction was defined using the Abrams-Griffith nomogram and urethral resistive index. A positive ice water test was defined as presence of uninhibited bladder contraction with instillation of 0C saline at 50 cc per minute up to a maximum of 250 cc. Detrusor instability was defined according to the International Continence Society criteria using room temperature saline instillation. RESULTS: When patients with neurological disease were excluded, a positive ice water test was found in 71% of subjects with bladder outlet obstruction (12 of 17), which was significantly higher (p <0.0005, Yates corrected chi-square test) than the 7% positive ice water test rate in nonobstructed subjects (3 of 44). Conversely, the incidence of positive detrusor instability was not statistically different between the patients with or without bladder outlet obstruction. Of the subjects with neurological disease 85% (42 of 50) had a positive ice water test. The incidence of a positive ice water test was only 5 to 9% in patients with storage lower urinary tract symptoms. CONCLUSIONS: A positive ice water test has been previously described in infants and individuals with neurogenic bladders. However, subjects with bladder outlet obstruction had a significantly higher incidence of a positive ice water test compared to those without it, supporting the hypothesis of an enhanced spinal micturition reflex possibly due to plasticity of bladder afferents after bladder outlet obstruction. The ice water test may be useful in prognosticating bladder outlet obstruction treatment outcomes and determining the etiology of treatment failure.

Evaluation of sexual behavior of hair sheep rams in a tropical environment.

We evaluated sexual behavior of St. Croix White (SC; n = 5) and Barbados Blackbelly hair (BB; n = 4) rams under two environmental conditions in the tropics. Sexually naive rams were individually exposed for 15 min to a restrained, ovariectomized ewe, three times during a 3-wk period in June, in a pen with shade (SHADE; 33.1+/-.3 degrees C) or without shade (SUN; 38.3+/-.3 degrees C). Rectal temperature (RT) of rams was measured before and after each test. Sexual behaviors were recorded by observers outside the pens. The number of mounts and ejaculations were similar (P > .10) between the SUN (12.1+/-2.8 and 3.6 +/-.5, respectively) and SHADE (10.7+/-2.9 and 3.4+/-.4, respectively) tests. There was no breed x test pen interaction for any of the behaviors recorded (P > .10). The BB rams mounted the ewe more (P < .04) than did the SC rams (15.7+/-2.8 vs 7.3+/-2.7 mounts, respectively). The overall level of activity (foreleg kicks, attempted mounts, mounts, and ejaculations) was similar (P > .10) between BB and SC rams (64.9 +/-8.5 vs 45.4+/-8.5 events, respectively). Rectal temperature before testing was similar (P > .10) in BB and SC rams (39.4+/-.1 vs 39.4+/-.1 degrees C, respectively). The change in RT of rams was not different (P > .10) between SUN and SHADE tests (.6 +/-.1 vs .8+/-.1 degrees C), but BB rams had a greater (P < .02) change in RT than SC rams (.9+/-.1 vs .5+/-.1 degrees C, respectively). The change in RT was positively correlated with time to first service (r = .39, P < .01) and number of mounts (r = .52, P < .001) and negatively correlated with number of services (r = -.47, P < .0008). These results show that under tropical conditions, hair sheep rams exhibit a full repertoire of sexual behaviors. There does not seem to be a negative influence of elevated ambient temperature during testing on the level of sexual behavior of these rams.