Swallowing Problems in Spinal Muscular Atrophy Types 2 and 3: A Clinical, Videofluoroscopic and Ultrasound Study.

BACKGROUND: Spinal muscular atrophy (SMA) is a hereditary motor neuron disorder, characterized by the degeneration of motor neurons and progressive muscle weakness. There is a large variability of disease severity, reflected by the classification of SMA types 1-4. OBJECTIVE: The aim of this cross-sectional study was to determine the nature of swallowing problems and underlying mechanisms in patients with SMA types 2 and 3, and the relationship between swallowing and mastication problems. METHODS: We enrolled patients (aged 13-67 years) with self-reported swallowing and/or mastication problems. We used a questionnaire, the functional oral intake scale, clinical tests (dysphagia limit, and timed test swallowing, the test of mastication and swallowing solids), a videofluoroscopic swallowing study (VFSS), and muscle ultrasound of the bulbar muscles (i.e. digastric, geniohyoid and tongue muscles). RESULTS: Non-ambulant patients (n = 24) had a reduced dysphagia limit (median 13 ml (3-45), and a swallowing rate at the limit of normal (median 10 ml/sec (range 4-25 ml). VFSS revealed piecemeal deglutition and pharyngeal residue. We found pharyngo-oral regurgitation in fourteen patients (58%), i.e. they transported the residue from the hypopharynx back into the oral cavity and re-swallowed it. Six patients (25%) demonstrated impaired swallowing safety (i.e. penetration aspiration scale > 3). Muscle ultrasound revealed an abnormal muscle structure of the submental and tongue muscles. Ambulant patients (n = 3), had a normal dysphagia limit and swallowing rate, but VFSS showed pharyngeal residue, and muscle ultrasound demonstrated an abnormal echogenicity of the tongue. Swallowing problems were associated with mastication problems (p = 0.001).

Mechanisms of haptoglobin protection against hemoglobin peroxidation triggered endothelial damage.

Extracellular hemoglobin (Hb) has been recognized as a disease trigger in hemolytic conditions such as sickle cell disease, malaria, and blood transfusion. In vivo, many of the adverse effects of free Hb can be attenuated by the Hb scavenger acute-phase protein haptoglobin (Hp). The primary physiologic disturbances that can be caused by free Hb are found within the cardiovascular system and Hb-triggered oxidative toxicity toward the endothelium has been promoted as a potential mechanism. The molecular mechanisms of this toxicity as well as of the protective activities of Hp are not yet clear. Within this study, we systematically investigated the structural, biochemical, and cell biologic nature of Hb toxicity in an endothelial cell system under peroxidative stress. We identified two principal mechanisms of oxidative Hb toxicity that are mediated by globin degradation products and by modified lipoprotein species, respectively. The two damage pathways trigger diverse and discriminative inflammatory and cytotoxic responses. Hp provides structural stabilization of Hb and shields Hb's oxidative reactions with lipoproteins, providing dramatic protection against both pathways of toxicity. By these mechanisms, Hp shifts Hb's destructive pseudo-peroxidative reaction to a potential anti-oxidative function during peroxidative stress.

Elucidating the role of matrix stiffness in 3D cell migration and remodeling.

Reductionist in vitro model systems which mimic specific extracellular matrix functions in a highly controlled manner, termed artificial extracellular matrices (aECM), have increasingly been used to elucidate the role of cell-ECM interactions in regulating cell fate. To better understand the interplay of biophysical and biochemical effectors in controlling three-dimensional cell migration, a poly(ethylene glycol)-based aECM platform was used in this study to explore the influence of matrix cross-linking density, represented here by stiffness, on cell migration in vitro and in vivo. In vitro, the migration behavior of single preosteoblastic cells within hydrogels of varying stiffness and susceptibilities to degradation by matrix metalloproteases was assessed by time-lapse microscopy. Migration behavior was seen to be strongly dependent on matrix stiffness, with two regimes identified: a nonproteolytic migration mode dominating at relatively low matrix stiffness and proteolytic migration at higher stiffness. Subsequent in vivo experiments revealed a similar stiffness dependence of matrix remodeling, albeit less sensitive to the matrix metalloprotease sensitivity. Therefore, our aECM model system is well suited to unveil the role of biophysical and biochemical determinants of physiologically relevant cell migration phenomena.

MAGE-C1/CT-7 expression in plasma cell myeloma: sub-cellular localization impacts on clinical outcome.

Plasma cell myelomas (PMs) have a poor prognosis. Cancer-testis (CT) antigens are immunogenic proteins, representing potential targets for tumor vaccination strategies. The expression of the CT antigens GAGE, MAGE-A4, MAGE-C1/CT-7, and NY-ESO-1 was investigated on paraffin-embedded bone marrow biopsies from 219 PM and 8 monoclonal gammopathy of undetermined significance (MGUS) patients. The frequency and prognostic impact of these CT antigens were compared with known morphological prognostic markers (i.e. Mib1 labeling index) and the presence of the translocations t(4;14)(p16.3; q32) and t(11;14)(q13;q32). We show that MAGE-C1/CT-7 is the most prevalent CT antigen, expressed in 57% of PMs in a high percentage of tumor cells. While MAGE-C1/CT-7 was absent in non-malignant plasma cells, plasma cells of patients with MGUS did express MAGE-C1/CT-7, but no other CT antigens. MAGE-C1/CT-7 was more frequently expressed in PMs with an elevated proliferation rate (Mib1 >10%) compared to PMs with a low proliferation rate (Mib1

High-resolution CryoFESEM of individual cell adhesion molecules (CAMs) in the glycocalyx of human platelets: detection of P-selectin (CD62P), GPI-IX complex (CD42A/CD42B alpha,B beta), and integrin GPIIbIIIa (CD41/CD61) by immunogold labeling and stereo imaging.

The aim of this study was to develop a model for the detection of individual cell adhesion molecules (CAMs) in the glycocalyx of spread human platelets using high-resolution cryo-field emission scanning electron microscopy (cryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPIba in the GPI-IX complex (CD42a/CD42b alpha,b beta), and the integrin GPIIbIIIa (CD41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled with 10-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coated unidirectionally with Pt, stabilized with carbon, and examined in an in-lens cryoFESEM using high-resolution backscattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type of CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of using high-resolution cryoFESEM to recognize and detect individual CAMs in the glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of stereo images), may provide better definition of the topography associated with glycosylation and formation of multimeric CAM complexes. (J Histochem Cytochem 49:809-819, 2001)

High-resolution backscatter electron detection of cell surface molecules on human platelets using the double-layer coating method and cryo field emission scanning electron microscopy.

A model system utilizing cryo scanning electron microscopy (SEM) for the detection of putative cell adhesion molecule(s) on the surface of human platelets is described. Plunge freezing was used for cryoimmobilization of unactivated and activated platelets after prefixation. Extracellular ice was removed by sublimation to expose the surface of the platelet membrane. Cryosamples were coated by the double-layer method, in which undirectional shadowing is performed at an angle of 45 degrees with 2 nm of platinum by thermal evaporation, followed by evaporation of 5 nm of carbon at an angle of 90 degrees for stabilization of the platinum film. The topography of the extracellular surface of the unstimulated platelet membrane was dominated by small spherical protrusions, while that of the activated platelet had not only similar spherical projections, but also possessed numerous rod-like protrusions, presumably representing the upregulation of the cell adhesion molecule, P-selectin, from intracellular a granules. These results clearly demonstrate that cryo field-emission SEM can detect molecular topography on the extracellular surface of cells consistent with the dimensions and shape of membrane cell adhesion molecules.

Quantitative immuno-gold labelling and ultrastructural preservation after cryofixation (combined with different freeze-substitution and embedding protocols) and after chemical fixation and cryosectioning. Analysis of the secretory organelle matrix of Paramecium trichocysts.

Among the variety of parameters affecting immuno-gold labelling efficiency, mainly the effects of different preparative protocols were tested. Preservation of ultrastructure and of antigenicity are the salient features of this study. We have labelled insoluble components of the secretory matrix of Paramecium trichocysts with specific antisera, using 10 nm colloidal gold particles. The highest labelling efficiency was obtained with fast freezing (cryofixation, either by sandwich or spray-freezing), freeze-substitution in methanol (without added fixatives) and hydrophilic Lowicryls, particularly when applied at low temperatures (K11M at 193 K). The presence of different chemical fixatives always reduced the labelling density and some recommendations from the literature do not appear advisable. Methods commencing with fixation at greater than or equal to 0 degree C, such as "progressive lowering of temperature" (PLT) or preparation of cryostat sections, i.e. with chemical pretreatments, always resulted in lower labelling density. Our data appear, therefore, relevant for optimal immuno-gold labelling of insoluble antigens and emphasize the potential of cryofixation as a primary preparation step. In addition, ultrastructural preservation was also superior after cryofixation.