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PubMed2000

Increased serum amyloid a levels reflect colitis severity and precede amyloid formation in IL-2 knockout mice.

The lack of sensitive and relatively non-invasive measures has hampered monitoring the clinical course of spontaneously developing colitis in IL-2-deficient (-/-) mice. We selected (i) to study the correlation of the acute phase plasma proteins serum amyloid A (SAA) and serum amyloid P component (SAP) levels with colonic disease and (ii) to characterize the amyloidosis in the IL-2(-/-)animals. IL-2(-/-)mice exhibited increasing severity of gross intestinal inflammation with age, confined to the distal colon. Histologically, the colonic disease score increased serially in IL-2(-/-)animals. Wild-type mice showed no activity, while 16-week-old IL-2(+/-)animals had minimal colitis with small ulcers and lamina propria inflammatory infiltrate. Periportal hepatitis was present and positive Congo red staining indicated amyloidosis of the liver and spleen in 16 week IL-2(-/-)mice. SAA immunostaining in the liver and spleen was increased in the 8 week and 16 week IL-2(-/-)and 16 week IL-2(+/-)animals indicating AA amyloid deposits. Plasma SAA and SAP levels were markedly elevated, and generally preceded the onset of colitis and reflected its severity. Northern analysis showed markedly increased SAA expression in the liver and intestine of IL-2(-/-)and intestine of IL-2(+/-)16-week-old animals. Increased intestinal expression of SAA3 (lamina propria macrophages) indicates local inflammation in IL-2(+/-)animals at 16 weeks. Treatment of 3-week-old animals with systemic IL-2 or IL-1 receptor antagonist (IL-1ra) delayed inflammation, postponed the increase in SAA levels and minimized disease onset. These results further demonstrate that IL-2 plays a significant role in normal immune responses in the body and that plasma SAA levels both reflect colonic disease severity and may indicate subclinical disease in both IL-2(-/-)and IL-2(+/-)mice. Furthermore. The mechanism of IL-2-deficient induced colitis appears to be mediated in part through the increase in IL-1. In addition, the IL-2(-/-)mouse of spontaneous enterocolitis may provide a unique system for studying spontaneously developing AA amyloidosis.

Amyloidosis↗

PubMed1997

Neutron and X-Ray Reflectivity Studies of the Adsorption of Aerosol-OT at the Air-Water Interface: The Structure of the Calcium Salt

We have used neutron and X-ray reflection to determine the structure of a layer of calcium bis-(2-ethyl 1-hexyl) sulphosuccinate (Aerosol-OT or AOT) adsorbed at the air/solution interface. The widths of the distributions of the chains and head groups of the molecule, and their positions in relation to the underlying water, have been measured at four concentrations varying from the solubility limit (CMC) at 4 x 10(-4) M to 1 x 10(-6) M. Over this concentration range the coverage changes from 68 ± 3 to 142 ± 8 Å2 per AOT unit. The structure of the layer both is quite different from that of NaAOT and varies quite differently with surface concentration. The Ca(AOT)2 layer is slightly (1 Å) further out from the water, but the chain region is thinner for the calcium surfactant. This is reflected most in the greatly reduced chain to head separation, which drops from about 6 Å in NaAOT to about 4 Å in Ca(AOT)2.

Journal Article↗

PubMed1997

Kinetic Analyses of the Colloidal Crystallization of Silica Spheres As Studied by Reflection Spectroscopy

Reflection spectroscopy is used in kinetic analyses of the nucleation and growth processes of colloidal crystals of silica spheres (110 nm in diameter) in exhaustively deionized aqueous suspensions. Sphere concentrations range from 0.001 to 0.0025 in volume fraction (phi) for the nucleation and 0.0014 to 0.0036 for the crystallization process, respectively. Induction periods are from 1 to 500 s and become longer with decreasing sphere concentration. Nucleation rates are 10(-3) to 10(3) &mgr;m-3 s-1 and increase sharply as sphere concentration increases. The crystallization process has been observed through the sharpening and the increase of intensity in the reflection peaks for the suspension in a test tube, which stands still after being inverted. Crystal growth rates v range from 2 to 15 &mgr;m/s and decrease linearly as the reciprocal sphere concentration increases. Crystal growth rates represented by the number of unit cells u also increase as phi increases, ranging from 2 to 23 unit cells/s. The importance of electrostatic intersphere repulsion through electrical double layers and of cooperative fluctuation of colloidal spheres in crystallization processes is supported. Copyright 1997Academic Press

Journal Article↗

PubMed1998

Kinetic Analyses of Colloidal Crystallization in Alcoholic Organic Solvents and Their Aqueous Mixtures As Studied by Reflection Spectroscopy.

Reflection spectroscopy is used for the kinetic analyses of the nucleation and growth process of colloidal crystals of silica spheres (110 nm in diameter) in exhaustively deionized suspensions of purely alcoholic organic solvents (methyl alcohol, ethyl alcohol, and ethylene glycol) and aqueous mixtures with alcohols (methyl, ethyl, n-propyl, and n-butyl alcohols and ethylene glycol). Sphere concentrations studied range from 0.001 to 0.01 in volume fraction, rather high compared with those in water. Induction periods are from 5 to 2000 s and are prolonged with decreasing sphere concentration. Nucleation rates are 10(-3) to 10(3) mm-3 s-1 and increase sharply as sphere concentration increases. The crystal growth rates, v have been determined from the increase of intensity in the sharpened reflection peaks. Values of v range from 1 to 27 µm/s and decrease linearly as the reciprocal sphere concentration increases. Nucleation and crystallization rates decrease sharply as the fraction of the organic solvents increases in the mixtures with water. The importance of the electrostatic intersphere repulsion through the electrical double layers and the cooperative and synchronized fluctuation of colloidal spheres in the crystallization processes is supported. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

The Denaturation of Lysozyme Layers Adsorbed at the Hydrophobic Solid/Liquid Surface Studied by Neutron Reflection.

We have studied the adsorption of lysozyme layers at a hydrophobic silicon water interface using specular neutron reflection. The hydrophobic surface was obtained by self-assembly of a densely packed monolayer of octadecyltrichlorosilane (OTS) onto the natural silica layer on the smooth surface of a (111) silicon block. The effect of pH on the adsorbed lysozyme layer was examined at a constant lysozyme concentration of 0.03 g dm-3 and at a constant ionic strength of 0.02 M. Reflectivity profiles at different pH show that adsorption is irreversible with respect to pH, the composition and structure of the final layer being dependent on the route by which the pH was achieved. The adsorbed protein layer was found to divide into approximately two regions, a densely packed thin layer next to the OTS surface and a diffuse thicker layer extending into the bulk solution. None of the dimensions of this structure corresponds to those of the globular protein in solution, suggesting that, unlike its adsorption at the hydrophilic silica/water interface, lysozyme is denatured at the OTS/water surface. The irreversible adsorption is explained by the combined interaction of the hydrophobic attraction of the hydrophobic fragments in lysozyme to the OTS surface and electrostatic repulsion within the adsorbed layer. The hydrophobic surface induces the exposure of hydrophobic fragments from the lysozyme assembly. The thickness of the dense layer suggests that the denatured protein adsorbs in the form of peptide chains with the hydrophobic amino acid side chains attached to the OTS surface with the hydrophilic side chains extending into the bulk solution. Since lysozyme is more stable at pH 7 than at pH 4, the difference in initial adsorption is dominated by the greater relative stability of lysozyme to denaturation at the higher pH. A change of pH from 7 to 4 reduces the stability of the protein to unfolding and results in more adsorption than when the pH is changed in the opposite direction. Solution pH also affects the net charges within the hydrophilic tail region and the structural distribution of the tail region was found to vary with pH. Copyright 1998 Academic Press.

Journal Article↗

PubMed1999

Kinetic Analyses of Colloidal Crystallization in a Sinusoidal Electric Field as Studied by Reflection Spectroscopy.

The effect of a sinusoidal electric field on crystal growth rates in the colloidal crystallization of silica spheres (110 nm in diameter) in exhaustively deionized aqueous suspensions has been studied by reflection spectroscopy. Sphere concentration is 0.0016 in volume fraction. Nucleation time is shorter than 1 s. The crystal growth rates, v, of the body-centered cubic lattices have been determined from the increase in the cube root of the intensity in the sharpened reflection peaks. The v value is 20 µm/s in the absence of an electric field. v decreases from 20 to 8 µm/s as the voltage applied increases from 0 to 10 V at 1 Hz. v decreases from 30 to 15 µm/s when frequency increases from 0.01 to 10 Hz at E = 6 V, and remains constant irrespective of frequencies higher than 10 Hz up to 10 kHz. Interestingly, the crystallization of bcc lattices is enhanced at low frequencies between 0.01 and 0.5 Hz. The main causes for the retardation of crystallization at high frequencies and voltages are (a) the additional translational fluctuation of the spheres and the surrounding electrical double layers by the electric field, and (b) the partial melting of the crystals by the shearing forces in an electric field. The importance of electrostatic intersphere repulsion resulting from overlap of the electrical double layers and cooperative and synchronized fluctuation of colloidal spheres in crystallization processes is strongly supported. Copyright 1999 Academic Press.

Journal Article↗

PubMed2001

Ellipsometry and Infrared Reflection Absorption Spectroscopy of Adsorbed Layers of Soluble Surfactants at the Air-Water Interface.

Optical techniques play an increasingly important role in the characterization of microstructure and surface densities of thin films at various interfaces. In this study, ellipsometry and infrared reflection absorption spectroscopy (IRRAS) were used for determining the surface densities of adsorbed layers of cationic surfactants in situ at the air-water interface. The surfactants were N(alpha)-lauroyl-arginine methyl ester (LAM) and N(alpha), N(omega)-bis(N(alpha)-lauroyl-arginine)-alpha,omega-alkylidenediamide (C(6)(LA)(2)). In ellipsometry, the ellipsometric phase angle Delta was obtained at various surfactant concentrations and was referenced to that of the solvent. Three algorithms were used for analyzing the data. The surface densities are 3.3+/-0.3x10(-6) mol/m(2) at 1 mM for LAM and 1.5+/-0.3x10(-6) mol/m(2) at 0.1 mM for C(6)(LA)(2) by using an algorithm for which the monolayer thickness was estimated from molecular modeling. The corresponding surface densities from literature surface tension data and the Gibbs adsorption isotherm procedure are 2.2+/-0.4x10(-6) mol/m(2) and 1.2+/-0.2x10(-6) mol/m(2), respectively. In addition, IRRAS spectra were obtained from monolayers of LAM and C(6)(LA)(2) at the air-water interface. The frequencies of the methylene stretching vibration bands indicate that the monolayers are liquid-like. The surface densities were determined from the reflectance-absorbance data by using the model of either an isotropic film or an anisotropic film on the aqueous subphase. The IRRAS-based surface densities from either model, by using DPPC monolayers for calibration, are 2.4+/-0.7x10(-6) mol/m(2) at 1 mM for LAM and 1.5+/-0.6x10(-6) mol/m(2) at 0.1 mM for C(6)(LA)(2), which are in fair agreement with the ellipsometry- and the surface-tension-based surface densities. Copyright 2001 Academic Press.

Journal Article↗

PubMed2001

Reflection spectroscopy of analgesized skin.

Analgesized skin, when subjected to heat stimuli, responds by increasing skin perfusion. This response does not originate from increased perfusion in superficial capillaries, but rather in the deeper lying vessels. The aim of this study was to assess changes in blood chromophore content, measured by reflection spectroscopy, in relation to the perfusion increase, especially regarding the chromophores oxyhemoglobin and deoxyhemoglobin. Eleven normal subjects were treated with analgesic cream (EMLA) and placebo for 20, 40, 60, 120, and 180 min. Individual reactions to local heating were classified as responses if the change in reflection data or the change in perfusion, as measured by laser Doppler blood flowmetry, exceeded 2 standard deviations of normal variation. The increase in blood perfusion or in blood content gave rise to an increased absorption, interpreted as an increase due mainly to the chromophore oxyhemoglobin. The number of responses increased with increased treatment time for EMLA-treated areas. In general, there was a good agreement between both methods; 44 of 55 classifications coincided for the two methods used. In conclusion, analgesized forearm skin, which had been exposed to local heating, responded with an elevated perfusion consisting of oxygenated blood. This strengthens the hypothesis that the flow increase occurs through dilatation of larger deeper lying skin vessels and not in the capillaries.

Adult↗

PubMed1995

Review: does measurement of regional cerebral blood flow reflect synaptic activity? Implications for PET and fMRI.

The energy metabolism of the adult human brain almost completely depends on glucose. The functional coupling of regional cerebral blood flow and local cerebral glucose metabolism has been established in a wide range of experiments using autoradiographic techniques in rats, cats, and monkeys as well as double-tracer techniques in humans. Glucose utilization in turn reflects neuronal activity and more specifically synaptic, mainly presynaptic, activity. The majority of glucose is needed for the maintenance of membrane potentials and restoration of ion gradients. PET as well as fMRI may be used to study changes in blood flow or flow-related phenomena in human subjects in vivo. Both techniques monitor changes of synaptic activity in a population of cells. These changes may be due to excitation or inhibition. More than 85% of cerebral glucose is used by neurons (mainly presynaptic axon terminals), while the remainder may at least partly account for metabolic processes in glial cells. Monitoring of regional cerebral blood flow with PET or fMRI thus mainly reflects neuronal and more specifically (pre-) synaptic activity.

Adult↗

PubMed1997

NIR reflection measurements of hemoglobin and cytochrome aa3 in healthy tissue and tumors. Correlations to oxygen consumption: preclinical and clinical data.

OBJECTIVES: A new near infrared reflectance spectroscopy based technology (MULTISCAN OS 10/30) for non-invasive measurements of tissue oxygenation allows detection of absolute tissue hemoglobin concentration and saturation values in real time. METHODS: MULTISCAN OS 10/30 scans a tissue sector of defined geometry at 400 to 1200 nm wavelength with 0.3 nm intervals at a scan rate of up to 400 Hz in reflection mode. The newly developed algorithms are based on the entropy pattern of photons and allow the detection of absolute values of total hemoglobin (tHb), deoxy- (Hb) and oxy-hemoglobin (HbO2) in mg/ml tissue as well as tissue oxygen (saturation = tiSO2) in the range of 0 to 100% in real time. Cytochrome aa3 (Cyt) can be monitored simultaneously. RESULTS: Physiological Stimulation. Clamping experiments at the finger of volunteers, in the intestine of a pig, in the kidney of mice and rats showed a very fast, sensitive and tissue specific reaction. Changes in breathing and/or anesthesia conditions were immediately followed by corresponding changes of tissue oxygenation (brain, finger, skin). Simultaneous measurements of oxygen (HbO2), and cytochrome showed a strong correlation between both parameters. For validation purposes parallel polarographic measurements (pO2 measurements) were performed in mice. Furthermore angiographic data of patients as well as NMR/I data were compared with the NIR spectroscopy findings. Tumor Measurements. Patients with tumors of different origins showed significantly different oxygen values. The lowest level was found in glioblastoma (< 10% sat, < 1.0 mg/ml tHb), whereas renal carcinoma showed tremendously increased values (> 90% sat, > 5.0 mg/ml tHb). In contrast the surrounding healthy kidney tissue was significantly less oxygenated and perfused compared to the tumors. Oxygen Consumption Measurements. Oxygen consumption was measured in 16 patients after ligation of the blood supply (A. and V. renalis) to the kidney affected by tumor. All tumors showed a significantly lower consumption rate compared to the healthy tissue. These findings were controlled by animal experiments of human renal carcinomas on nude mice. The same results were obtained under these experimental conditions. CONCLUSION: MULTISCAN OS 10/30 is a new and useful tool for in vivo characterization of oxygen and cytochrome in healthy and tumor tissues. In many clinically relevant situations oxygen measurements can be helpful and support the clinical routine diagnostics.

Adult↗

PubMed1997

Ca2+ sparks within 200 nm of the sarcolemma of rat ventricular cells: evidence from total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy (TIRFM) was used to measure local calcium releases in resting cardiac myocytes stained with fluo-3AM. The measured fluorescence originated from regions where cells were close to, and develop adhesions to, a totally reflecting glass surface. The excitation of the fluorescent Ca2+ indicator dye by the exponentially attenuated evanescent wave penetrated approximately 200 nm into the fluid phase. In rat ventricular cells, Ca2+ waves and Ca2+ sparks were observed within the adhesions. Ca2+ sparks recorded with TIRFM compared favorably to sparks recorded under similar conditions with confocal microscopy. Computer simulation supported this assessment. It is concluded that TIRFM can provide an economical, flexible tool for detailed measurement of Ca(2+)-transients in the subsarcolemmal space of live cells.

Animals↗

PubMed1998

Studies on the 14.5 nm meridional X-ray diffraction reflection during length changes of intact frog muscle fibres.

The intensity of the 14.5 nm meridional reflection (M3) from activated skeletal muscle fibres was studied in both single fibres and fibre bundles during the imposition of length changes. During shortening at small load, the intensity of the reflection decreased within 2 ms to less than 20% of isometric intensity, then recovered partially during the remainder of the shortening. When shortening was terminated, recovery of intensity was delayed. Small shortening steps (0.5% fibre length) produced a fall in M3 intensity (IM3) delayed by ca. 250 microseconds compared to the fall in tension. For larger step releases (1% fibre length), the fall in IM3 was not delayed. The fall in IM3 could be almost completely reversed by a subsequent restretch applied within 1.5 ms. Beyond 10 ms after the initial release, the restretch caused a further fall in intensity. A rapid step stretch (0.5% fibre length) also caused a fall in IM3 without delay, which was partially reversed by a release applied within 10 ms. A second small release applied 3 ms (or less) after the first caused a second fall in M3 intensity, but without delay and with faster time course. Small amplitude sinusoidal length oscillations (0.15-0.2% sarcomere; 1 kHz) caused a sinusoidal change in M3 intensity, which was 180 degrees out of phase with the force oscillations, and lacked distortion during its release phase.

Animals↗

PubMed2006

Reflection contrast microscopy: The bridge between light and electron microscopy.

Reflection contrast microscopy (RCM) is a light microscopic method to image cells at high definition and enhanced sensitivity compared to conventional bright-field microscopy. RCM images have very high contrast, which makes them easily applicable for digital image analysis. Because ultrathin sections are mostly used in this method, RCM also functions by bridging light with electron microscopy: the combination of ultrastructural with histochemical studies. RCM can also replace electron microscopy for rapid and simple screening of large quantities of samples for immunocytochemical staining. Special attention is paid to small biological objects, which have to be processed for RCM. If you encounter the limits of brightfield microscopy, in resolution, sensitivity or handling of the specimen, RCM will be a feasible option. Reflection contrast microscopy methods use only slightly adjusted electron microscopy methods for specimen preparation. Therefore, many familiar techniques for ultrathin specimen preparation can be applied. It is essential that only refractive index differences exist in those areas that are of interest and that the further specimen is as optically homogenic as possible, with a refractive index as close to that of glass as possible. Therefore, plastic embedding is recommended.

Animals↗

PubMed1990

Reflection of changes in membrane constituents in various regions of Alzheimer brains to differential scanning thermograms.

To test whether changes in basic biochemical membrane constituents were reflected in membrane fluidity measurements, protein, total lipids, triglycerides, lecithin, cholesterol and alkaline phosphatase were determined in frontal cortex, hippocampus, putamen and nucleus basalis Meynert (NbM) of DAT brains and controls and compared to differential scanning thermograms. Biochemical changes were most pronounced in the hippocampus, while thermostability was altered in the NbM and the frontal cortex. The results indicate that changes in protein content, but not in lipid composition were reflected in alterations of thermostability of the brain regions examined.

Aged↗

PubMed1995

Combined analysis of in situ hybridization, cell cycle and structural markers using reflectance and immunofluorescence confocal microscopy.

A method for the simultaneous detection of mRNA by reflectance in situ hybridization (RISH), cell cycle and structural markers by immunofluorescence using confocal laser scanning microscopy is presented. The mRNA expression of two ras-related genes rhoB and rhoC was analysed in human breast cancer cell lines and human histological specimens (breast cancer tissues and skin biopsies). In breast cancer cell lines, the conditions were optimized to detect RNA-RNA hybrids and DNA synthesis after pulse-labelling with bromodeoxyuridine. Endonuclease-exonuclease digestion, which allows the accessibility to specific antibodies of halogenated pyrimidine molecules, was carried out following ISH. Finally, cytokeratin or vimentin staining was performed. The detection of signals, arising from 1-nm colloidal gold particles without silver enhancement, by reflectance confocal laser scanning microscopy is described. Bromodeoxybiridine DNA markers and cytokeratin/vimentin staining were detected concomitantly using different fluorochromes. To allow comparative expression of two related genes, the mRNA of rhoB and rhoC were detected using digoxigenin- or biotin-labelled riboprobes and, after 3-D imaging, a detailed analysis by optical horizontal (x, y) and vertical (x, z) sectioning was undertaken. The subsequent bromodeoxyuridine detection procedure permitted to us explore the specific transcription of these two genes during S and non-S phases. This method allows the identification and localization of several subcellular components in cells within a complex tissue structure and makes it possible to analyse further transcript localization in relation to the function of the encoded protein and to the cell cycle.

Biomarkers↗

PubMed1976

Electron microscopy of two types of reflecting chromatophores (iridophores and leucophores) in the guppy, Lebistes reticulatus Peters.

Reflecting chromatophores in the integument of the guppy, Lebistes reticulatus Peters, are of two distinct types, iridophores and leucophores. The iridophores are smaller and fixed, producing a metallic iridescent color. The cytoplasmic organelles involved in the coloration of iridophores are the reflecting platelets, as in the iridophores of other fish and amphibian species on which earlier reports have been made. Spherical granules of pleiomorphic internal structure, quite variable in size but generally 0.2 mum to 1.0 mum in diameter, are also numerous in the iridophores. The nature of these granules remains unknown. The leucophores are larger, and highly dendritic; their pigment granules are migratory and they exhibit a dull whitish color. Pigment granules of the leucophores are spherical in form, varying from 0.5-0.8 mum in diameter, with a double membrane enclosing the internal fibrous materials. Melamine-treatment of the fish caused degenerative changes in the pigment granules and also the other cytoplasmic organelles of the leucophores, whereas the other kinds of chromatophores, including the iridiophores, remained intact. Some problems in general characterization and classification between these two types of chromatophores were discussed.

Animals↗

PubMed1993

Multicolour preparations for in situ hybridization using precipitating enzyme cytochemistry in combination with reflection contrast microscopy.

We have further developed a method for the detection of different enzyme cytochemical reaction products by means of reflection contrast microscopy (RCM). By embedding these enzyme precipitates in a protein matrix, we were able to prevent the reaction products from dissolving in immersion oil, which is required for RCM analysis. The applicability of the RCM procedure is, therefore, extended to a range of cytochemical enzyme precipitation methods, which normally result in oil soluble reaction products. To test their usefulness, these enzyme precipitates have been used in single- and well as double-label in situ hybridization (ISH) procedures to visualize a number of DNA target sequences by several different reflection colours, i.e. white, yellow and red. Three repetitive DNA probes for the (sub)centromeric regions of chromosomes 1, 7 and 17, as well as a repetitive DNA probe for the telomeric region of chromosome 1, and two cosmid DNA probes (40 kb each) for both arms of chromosome 11 could be detected with high efficiency in both interphase and metaphase preparations. Moreover the enzyme precipitates were shown to be stable upon exposure to excitation light or upon storage. It may be concluded that these findings render RCM a sensitive method for the visualization of multiple targets in biological specimens.

Carcinoma, Transitional Cell↗

PubMed1994

Reflection contrast microscopy within chrome-alum haematoxylin stained thick tissue-sections.

This paper introduces two innovations in reflection contrast microscopy (RCM): (1) an extended application for qualitative light microscopic investigations; and (2) a novel method for quantification in cytochemistry. (1) We found out that RCM cannot only be used for surface characterizations and in thin sections but also within thick tissue-sections. The use of the RCM technique is demonstrated on slides of the supraoptic nucleus (SON) of the rat stained with chrome-alum haematoxylin: Among all the stained structures only neurosecretory granules are found to cause reflections. The visualization of the neurosecretion and its distribution is more distinct and of sharper contrast than in bright field microscopy. (2) The improved differentiation allows the quantification of neurosecretion in tissue-sections by combining RCM with grey-scale image analysis.

Animals↗