[Paucisymptomatic presentation of an allergic bronchopulmonary aspergillosis]. / Une aspergillose broncho-pulmonaire allergique de présentation pauci-symptomatique.

Clinical atypical presentations of rare lung disease can cause a diagnostic issue. In this case report, allergic bronchopulmonary aspergillosis is not like in the traditional form of severe asthma but is revealed by a persistent cough. CT scan and biological examinations should be prescribed when there is doubt in confirming the diagnosis. Diagnostic and therapeutic management should be done early in this disease to avoid complications such as bronchiectasis.

[Severe hypercalcemia revealing sarcoidosis precipitated by etanercept]. / Hypercalcémie majeure révélatrice d'une sarcoïdose induite par étanercept.

INTRODUCTION: The principal secondary effects of anti-TNF alpha therapy are now well understood, particularly the risk of opportunistic infections. Other paradoxical effects have been described much more occasionally such as the developement of sarcoid-like granulomatous reactions. CASE REPORT: We report here the case of a woman of 39 years treated for severe rheumatoid arthritis for five years with etanercept. She was admitted to hospital as an emergency with vomiting and diffuse abdominal pain. Investigations revealed severe hypercalcaemia and acute renal failure. After correction of the metabolic disturbances with rehydration and biphosphonates, CT scanning of the abdomen, pelvis and thorax showed bilateral interstitial infiltration and splenomegaly. The diagnosis of sarcoidosis was confirmed by endoscopic bronchial biopsies. Progress was satisfactory following withdrawal of the etanercept and corticosteroid therapy in reducing dosage. CONCLUSION: The risk of induced sarcoidosis should be understood in patients receiving anti-TNF therapy and should be considered in cases of hypercalcaemia and/or splenomegaly.

A dedicated two-element phased array receiver coil for high resolution MRI of rat knee cartilage at 7T.

Despite that on clinical systems phased array technology is now widely used, the high field MRI experimental systems with multiple receiver channels just became available few years ago. For this reason and due to the large range of magnetic field (frequencies between 200 and 500 MHz for proton resonance), commercial phased arrays implemented in narrow bore for high field applications are rare and relatively expensive. Array coil imaging is an advanced method for acquiring high resolution images with enhanced Signal-to-Noise Ratio (SNR) and/or enlarged Field Of View (FOV) compared for example to single loop surface coil. The volume of interest is then covered by several coil elements and images reconstructed for every single channel are combined afterwards. The goal of this work was to develop a dedicated two-element array coil operating at 300 MHz (7T) for high-resolution imaging of rat knee joint in order to quantify cartilage thickness and volume. A dedicated two-element array coil with two square elements encompassing knee joint was designed and built. Decoupling between elements was achieved with a capacitor inserted on the common leg of the two elements. The average gain in SNR compared to a 15 mm reference single loop coil was 2.2. This SNR gain was used to improve spatial resolution of 3D acquisition by decreasing the voxel size from 59 x 59 x 156 microm(3) to 51 x 51 x 94 microm(3) without time penalty.

BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel.

OBJECTIVE: According to recent reports, the synovial membrane may contain mesenchymal stem cells with the potential to differentiate into chondrocytes under appropriate conditions. In order to assess the usefulness of synovium-derived progenitor cells for the purposes of cartilage tissue engineering, we explored their requirements for the expression of chondrocyte-specific genes after expansion in vitro. DESIGN: Mesenchymal progenitor cells were isolated from the synovial membranes of bovine shoulder joints and expanded in two-dimensions on plastic surfaces. They were then seeded either as micromass cultures or as single cells within alginate gels, which were cultured in serum-free medium. Under these three-dimensional conditions, chondrogenesis is known to be supported and maintained. Cell cultures were exposed either to bone morphogenetic protein-2 (BMP-2) or to isoforms of transforming growth factor-beta (TGF-beta). The levels of mRNA for Sox9, collagen types I and II and aggrecan were determined by RT-PCR. RESULTS: When transferred to alginate gel cultures, the fibroblast-like synovial cells assumed a rounded form. BMP-2, but not isoforms of TGF-beta, stimulated, in a dose-dependent manner, the production of messenger RNAs (mRNAs) for Sox9, type II collagen and aggrecan. Under optimal conditions, the expression levels of cartilage-specific genes were comparable to those within cultured articular cartilage chondrocytes. However, in contrast to cultured articular cartilage chondrocytes, synovial cells exposed to BMP-2 continued to express the mRNA for alpha1(I) collagen. CONCLUSIONS: This study demonstrates that bovine synovium-derived mesenchymal progenitor cells can be induced to express chondrocyte-specific genes. However, the differentiation process is not complete under the chosen conditions. The stimulation conditions required for full transformation must now be delineated.

Effect of articular cartilage proteoglycan depletion on high frequency ultrasound backscatter.

OBJECTIVE: To study the effect of variations of articular cartilage proteoglycans (PG) on high-frequency ultrasound backscatter. DESIGN: The study was performed on patellar cartilages of immature and mature rats (N=36). The variation of PG content was induced by enzyme digestion. Control and treated cartilages were explored in vitro using a 55MHz scanning acoustic microscopy, then assessed by histology for the fibrillar collagen organization analysis. The variations of proteoglycan and collagen content were evaluated. Thickness measurements performed on both B-scan images and histologic sections were compared. Ultrasonic radio-frequency signals reflected by the cartilage surface and backscattered from its internal matrix were processed to estimate the integrated reflection coefficient (IRC) and apparent integrated backscatter (AIB). RESULTS: Although hyaluronidase treatment of immature and mature cartilages removed approximately 50% of the proteoglycans, the echogenicity level of ultrasound images of degraded cartilages was similar to that of controls. IRC and AIB parameters did not significantly vary. Histologic sections of degraded cartilage displayed no change in collagen fiber organization. The thickness mean values measured by ultrasound in PG-depleted groups were significantly higher than in controls, whereas no significant difference in thickness was detected by histological measurement. The increase in cartilage thickness may potentially be explained by a decrease of speed of sound in PG-depleted cartilages that is more likely subsequent to an increase of water content. CONCLUSION: Current results indicate that PG depletion has no significant effect on high frequency ultrasound backscattered from rat patellar cartilage. Ultrasound may provide information about variations of PG content via speed of sound measurement.

Structural evaluation of articular cartilage: potential contribution of magnetic resonance techniques used in clinical practice.

OBJECTIVE: To determine whether routine magnetic resonance imaging (MRI) techniques can detect age-related structural modifications of bovine articular cartilage. METHODS: The cartilage of 3-month-old, 3-year-old, and 13-year-old animals was studied. T1- and T2-weighted MR sequences were performed using a 1.5T clinical imager and a 3-inch surface coil. Histologic slices (5 microm) of cartilage specimens were stained with picrosirius red (for collagen) and toluidine blue (for glycosaminoglycans [GAGs]). A polarized light study was performed to determine the collagen network organization. Except for the 13-year-old animal cartilage, the biochemical content was studied on slices cut parallel to the surface to determine GAG and hydroxyproline (collagen) content. Cartilage profiles were performed to determine the MR pixel intensity and the histologic color intensity. RESULTS: On T1-weighted images, the cartilage was homogeneous, with pixel intensity profiles presenting low variations. On T2-weighted images, the cartilage was laminar in the 3-month-old animals and became homogeneous thereafter. The pixel intensity varied through the cartilage depth with a profile that depended on the age of the animal. The collagen and GAG staining showed abrupt transitions in the 3-month-old animal, while in older animals the cartilage became more homogeneous with a mild gradient of matrix constituents with depth. These results were confirmed by findings of a biochemical study. In addition to these matrix content variations, the bovine cartilage presented modifications of its collagen network organization with aging. CONCLUSION: The MR T2-weighted sequences depicted signal variations with age in bovine cartilage concomitant with modifications in its structure. If confirmed in clinics, these observations will reinforce the place of MRI in characterizing cartilage with aging and pathologic processes.

T2 mapping of rat patellar cartilage.

PURPOSE: To investigate the usefulness of magnetic resonance (MR) T2 mapping in characterizing the evolution of cartilage matrix content and thickness during the maturation and aging process. MATERIALS AND METHODS: Patellae from four groups of rats aged 4 weeks, 8 weeks, 4 months, and more than 6 months ("old rats") were studied ex vivo with an 8.5-T microimager. T2 values were calculated on transverse rat patellar sections and displayed with a color scale (the T2 map) on a pixel-by-pixel basis. Biochemical and histologic studies were performed to evaluate the influence of proteoglycans and collagen contents on T2 values of the patellar cartilage. RESULTS: On the T2 map, the maturation process until 10 weeks was characterized by a decrease in T2 values and in cartilage thickness. The biochemical data revealed a global decrease in proteoglycans and a progressive global increase in collagen content, whereas the histologic study revealed subtle zonal variation in matrix constituents with depth. As aging progressed, the T2 values were low, without important variations, whereas the global cartilage thickness decreased. The cartilage matrix became globally more fibrotic, especially in the deepest zone. Biochemical analysis revealed that collagen content was more determinant of MR signal intensity than was proteoglycans content during maturation and aging. CONCLUSION: T2 mapping allows characterization of variations in cartilage matrix constituents and thickness.

Chloride currents in primary cultures of rabbit proximal and distal convoluted tubules.

Cl- conductances were studied in cultured rabbit proximal convoluted tubule (PCT) epithelial cells and compared with those measured in cultured distal bright convoluted tubule (DCTb) epithelial cells. Using the whole cell patch-clamp technique, three types of Cl- conductances were identified in DCTb cultured cells. These consisted of volume-sensitive, Ca2+-activated, and forskolin-activated Cl- currents. In PCT cultured cells, only volume-sensitive and Ca2+-activated Cl- currents were recorded. The characteristics of Ca2+-activated currents in PCT cells closely resembled those in DCTb cells. Volume-sensitive Cl- currents could be elicited both in PCT and in DCTb cells by hypotonic stress. The pharmacological profile of this conductance was established for both cell types. Forskolin activated a linear Cl- current in DCTb cells but not in PCT cells. This conductance was insensitive to DIDS and corresponds to cystic fibrosis transmembrane conductance regulator (CFTR)-like channels. Quantitative measurements of SPQ fluorescence showed that only the apical membrane of DCTb cells possessed a Cl- pathway that was sensitive to forskolin. RT-PCR experiments showed the presence of CFTR mRNA in both cultures, whereas immunostaining experiments revealed the expression of CFTR in DCTb cells only. The physiological role of the different types of channels is discussed.

Distribution of ionocytes in the saccular epithelium of the inner ear of two teleosts (Oncorhynchus mykiss and Scophthalmus maximus).

The saccular membranes of trout (Oncorhynchus mykiss) and turbot (Scophthalmus maximus) were examined to characterize specialized epithelial cells that might be responsible for ion exchange. The approach for localizing cell types was new for this tissue, as observations were made with a stereomicroscope and a light microscope in order to have a general view of the epithelium. No important differences between the two species were seen. The saccular tissue is a monolayer epithelium (except for the macula neural zone) surrounded by a layer of connective tissue invaded by many blood vessels. The use of the fluorescent probe DAPSMI and zinc iodide/osmium fixation-coloration defined two areas in which ionocytes were present. In the first, large ionocytes were grouped into a nearly complete, crowned meshwork around, but separated from, the macula. In the second area, opposite the macula, the ionocytes were smaller, cubical, and grouped in patches. Cells rich in Na+, K+-ATPase and carbonic anhydrase II were present in both areas. Contrary to previous studies in mammals and fish, ionocytes were also found in the epithelium of the saccule.

Ionic composition of endolymph in teleosts: origin and importance of endolymph alkalinity.

Ionic (Na+, K+, Cl-, PO4(3-), pH), total CO2, total calcium and protein concentrations in the plasma and endolymph of the inner ear were compared in trout Oncorhynchus mykiss and turbot Scophthalmus maximus. In both species, saccular endolymph was characterized by high levels of K+ and total CO2 and in trout by an alkaline pH. The kinetic characteristics of proton secretion across the saccular epithelium of trout were investigation using a titration technique in which isolated saccules were mounted as closed sacs. The rate of proton secretion depends strongly on the pH of the Ringer's solution and secretion stops at a pH below 7.2. Proton secretion is driven by an energy-dependent mechanism involving basolateral ouabain-sensitive Na+/K+ exchangers. Proton secretion was partially inhibited by acetazolamide and completely inhibited in Na(+)-free Ringer or in the presence of 1 mmol l-1 amiloride. A cellular model stressing the importance of proton exchange through the saccular epithelium is proposed to explain the regulation of endolymph pH, a crucial factor for the deposition of otolith calcium.

Simultaneous recognition of ionocytes and mucous cells in the gill epithelium of turbot and in the rat stomach.

A simple and rapid method is described which allows the recognition of different types of epithelial cells in one paraffin section. In the fish gill, the "chloride cells" (also called ionocytes or mitochondria-rich cells) appeared black and well contrasted after the zinc iodideosmium (ZIO) fixation, a contrast which is preserved after staining with alcian blue. All mucous cells appeared pale beige and can be clearly distinguished from the other cells but the known specificity of alcian blue for acid and sulfate mucins was lost. A complex tissue like the gill showed an interesting metachromasia. The combined ZIO fixation and alcian blue staining has also been applied on another epithelium, the rat stomach.

Ca(2+)-ATPase and Mg(2+)-ATPase in Aplysia glial and interstitial cells: an EM cytochemical study.

The localization of Ca(2+)- and Mg(2+)-ATPases was determined in Aplysia central and peripheral nervous system, using an electron microscopic cytochemical method. The enzyme activity appeared localized to the membrane of glial granules (gliagrana), particularly in the peripheral nervous system of the esophagus, and on the plasma membrane of central glial cells adjacent to neuronal cell bodies. No calcium- and/or magnesium-ATPase activity was detectable on the plasma membrane of glial cells surrounding nerve axons in the pleuro-visceral connectives. These findings are discussed along two main lines: (a) the calcium-ATPase of the gliagrana coincides with a high intragranular calcium and/or proton concentration; and (b) the presence of a calcium-ATPase activity at the glio-neuronal interface around the neuronal cell bodies coincides with the use of calcium ions as charge carriers of the action potential, and its absence at the level of the axon with the concurrent functional use of sodium ions.

Reconstituted skin in culture: a simple method with optimal differentiation.

Human skin is a unique organ, which can be reconstituted in vitro and represents an interesting system for studying cell proliferation and differentiation. A simple technique for producing reconstituted skin with optimal epidermal differentiation is described and characterized. A 4-mm punch biopsy of normal human skin is deposited on the epidermal side of mortified de-epidermized human dermis maintained at the air-liquid interface with a metallic support. The culture medium contains insulin, epidermal growth factor (EGF), cholera toxin, hydrocortisone, penicillin/streptomycin and fungizone. A well-differentiated epidermis develops within 15 days. Morphological and ultrastructural studies show a neoepidermis resembling normal skin. Differentiation markers such as involucrin, filaggrin, and various cytokeratins detected with pancytokeratin antibody are present and confirm this resemblance. The keratin profile is comparable to that observed in other skin culture models. A basement-membrane-like structure is reconstituted with hemidesmosomes and anchoring-filament formation. Bullous pemphigoid (BP) antigen is observed at the dermo-epidermal junction after 21 days of culture. Moreover, both dermal substrates and punch biopsies can be kept frozen for long-term storage, with little or no loss of epidermal growth kinetics and morphology. This skin culture technique is rapid, simple, economical and reproducible. Characterization has here shown high-quality epidermal differentiation. Scientists interested in epidermal in vitro studies should take interest in all these advantages.

Microinjection of p34cdc2 kinase induces marked changes in cell shape, cytoskeletal organization, and chromatin structure in mammalian fibroblasts.

We have examined the effects of elevating the intracellular levels of p34cdc2 kinase by microinjection into living mammalian cells. These studies reveal rapid and dramatic changes in cell shape with cells becoming round and losing the bulk of their cell-substratum contact. Such effects were induced at all times in the cell cycle except at S phase and were fully reversible at S phase or mitosis. Similar results were obtained with the homogeneous catalytic subunit of p34cdc2 kinase or p34cdc2 kinase associated with cyclin B. These alterations were accompanied by a marked reduction in interphase microtubules without the spindle formation, actin microfilament redistribution, and premature chromatin condensation. Although these changes closely mimic the events occurring during early phases of mitosis, p34cdc2 kinase-injected cells were not induced to pass further into division. These data provide detailed evidence that p34cdc2 kinase plays a major prerequisite role in the rearrangement of cellular structures associated with mammalian cell mitosis.

High-molecular-mass proteins in haemodialysis-associated amyloidosis.

1. Protein constituents were determined in eight amyloid deposits from eight patients (five male and three female), 53 +/- 4 years of age, treated by haemodialysis for 9-20 years using only cuprophane membranes and operated for carpal tunnel syndrome. 2. Soluble proteins were removed by solubilization in phosphate-buffered saline after osmotic lysis. The proteins of the insoluble fibrils were characterized by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and two-dimensional gel electrophoresis, and immunologically identified by Western blotting. 3. In addition to beta 2-microglobulin, alpha 2-macroglobulin was identified in the fibrillar material. The presence of these two proteins in amyloid deposits was confirmed by immunofluorescent microscopic studies. 4. Our data confirm the presence of beta 2-microglobulin in haemodialysis-associated amyloidosis, and also suggest a possible role for alpha 2-microglobulin: it may protect beta 2-microglobulin from proteolytic digestion, leading to its accumulation in intact form and to amyloid fibril formation.

A 36 kDa monomeric protein and its complex with a 10 kDa protein both isolated from bovine aorta are calpactin-like proteins that differ in their Ca2+-dependent calmodulin-binding and actin-severing properties.

Interaction of plasma membrane with the cytoskeleton involves a large number of proteins, among them a 36 kDa protein that was found to be involved in the interaction with actin filaments. We have isolated a 36 kDa protein from bovine aorta as a monomer and in a complex with a 10 kDa protein. Partial amino acid sequence determinations show that the 36 kDa and 10 kDa proteins isolated from bovine aorta are analogous to or identical with corresponding proteins purified from bovine intestine already described by Kristensen, Saris, Hunter, Hicks, Noonan, Glenney & Tack [(1986) Biochemistry 25, 4497-4503]. We report here that the association of the 10 kDa protein with the 36 kDa protein confers specific calmodulin-binding and actin-severing properties on the complex that are not possessed by the 36 kDa monomer alone. These findings suggest that the protein complex could be involved in thin-filament-related structures or could modulate some Ca2+-regulated events mediated by calmodulin.

[Sites of aluminum accumulation in bone marrow: study using electron microscopy, ionic microscopy and X-ray microanalysis]. / Sites d'accumulation de l'aluminium dans la moelle osseuse: étude par microscopie électronique microscopie ionique et microanalyse X.

Two methods of analytical microscopy have been used to study the distribution of aluminum in bone marrow of rats intoxicated by aluminum gluconate. Images of the distribution of aluminum in a field of 250 microns in diameter were obtained by analytical ion microscopy. They show that this element was concentrated in spots, associated with iron or alone, in the cytoplasm of some cells. Electron Probe Microanalysis (EPMA) has shown that aluminum concentration occurred in cells of the reticulo-endothelial system, principally in the reticular cells of erythroblastic islets. In cells of the reticuloendothelial system, aluminum was observed in intracytoplasmic organelles having ultrastructural characteristics of lysosomes or phagolysosomes. In these organelles, aluminum is always associated with phosphorus and sometimes with iron. No cytoplasmic or nuclear aluminum accumulation was detected in any other variety of bone marrow cells. The consequences of the selective accumulation of aluminum in the cytoplasm of reticular cells of erythroblastic islets for the maturation of erythrocytes are discussed.