Diagnosing eosinophilic asthma using a multivariate prediction model based on blood granulocyte responsiveness.

BACKGROUND: The identification of inflammatory asthma phenotypes, using sputum analysis, has proven its value in diagnosis and disease monitoring. However due to technical limitations of sputum analysis, there is a strong need for fast and noninvasive diagnostics. This study included the activation state of eosinophils and neutrophils in peripheral blood to phenotype and monitor asthma. OBJECTIVES: To (i) construct a multivariable model using the activation state of blood granulocytes, (ii) compare its diagnostic value with sputum eosinophilia as gold standard and (iii) validate the model in an independent patient cohort. METHODS: Clinical parameters, activation of blood granulocytes and sputum characteristics were assessed in 115 adult patients with asthma (training cohort/Utrecht) and 34 patients (validation cohort/Oxford). RESULTS: The combination of blood eosinophil count, fractional exhaled nitric oxide, Asthma Control Questionnaire, medication use, nasal polyposis, aspirin sensitivity and neutrophil/eosinophil responsiveness upon stimulation with formyl-methionyl-leucyl phenylalanine was found to identify sputum eosinophilia with 90.5% sensitivity and 91.5% specificity in the training cohort and with 77% sensitivity and 71% specificity in the validation cohort (relatively high percentage on oral corticosteroids [OCS]). CONCLUSIONS: The proposed prediction model identifies eosinophilic asthma without the need for sputum induction. The model forms a noninvasive and externally validated test to assess eosinophilic asthma in patients not on OCS.

The use of two different MLPA kits in 22q11.2 deletion syndrome.

22q11.2 deletion syndrome (22q11DS) is one of the most common recurrent copy-number variant disorder, caused by a microdeletion in chromosome band 22q11.2 and occurring with a population prevalence of 1 in 2000. Until today there has been no evidence that the size of the deletion has an influence on the clinical phenotype. Most studies report that 22q11DS is associated with mild or borderline intellectual disability. There are a limited number of reports on 22q11DS subjects with moderate or severe intellectual disability. In this study we describe 63 adult patients with 22q11DS, including 22q11DS patients functioning at a moderate to severe intellectual disabled level. Deletion size was established with an experimental Multiplex ligation-dependent probe amplification (MLPA) mixture (P324) in addition to the commonly used MLPA kit (P250). We compared deletion size with intellectual functioning and presence of psychotic symptoms during life. The use of the experimental MLPA kit gives extra information on deletion size, only when combined with the common MLPA kit. We were able to detect eleven atypical deletions and in two cases the deletion size was shorter than all other "typical ones". We conclude that the use of the experimental kit P324 gives extra information about the deletion size, but only when used together with the standard P250 kit. We did not found any relation of deletion size with intelligence or presence of psychosis.

Elemental mapping in achromatic atomic-resolution energy-filtered transmission electron microscopy.

We present atomic-resolution energy-filtered transmission electron microscopy (EFTEM) images obtained with the chromatic-aberration-corrected FEI Titan PICO at the Ernst-Ruska Centre, Jülich, Germany. We find qualitative agreement between experiment and simulation for the background-subtracted EFTEM images of the Ti-L2,3 and O-K edges for a specimen of SrTiO3 oriented down the [110] zone axis. The simulations utilize the transition potential formulation for inelastic scattering, which permits a detailed investigation of contributions to the EFTEM image. We find that energy-filtered images of the Ti-L2,3 and O-K edges are lattice images and that the background-subtracted core-loss maps may not be directly interpretable as elemental maps. Simulations show that this is a result of preservation of elastic contrast, whereby the qualitative details of the image are determined primarily by elastic, coherent scattering. We show that this effect places a constraint on the range of specimen thicknesses which could theoretically yield directly useful elemental maps. In general, interpretation of EFTEM images is ideally accompanied by detailed simulations.

Refinement procedure for the image alignment in high-resolution electron tomography.

High-resolution electron tomography from a tilt series of transmission electron microscopy images requires an accurate image alignment procedure in order to maximise the resolution of the tomogram. This is the case in particular for ultra-high resolution where even very small misalignments between individual images can dramatically reduce the fidelity of the resultant reconstruction. A tomographic-reconstruction based and marker-free method is proposed, which uses an iterative optimisation of the tomogram resolution. The method utilises a search algorithm that maximises the contrast in tomogram sub-volumes. Unlike conventional cross-correlation analysis it provides the required correlation over a large tilt angle separation and guarantees a consistent alignment of images for the full range of object tilt angles. An assessment based on experimental reconstructions shows that the marker-free procedure is competitive to the reference of marker-based procedures at lower resolution and yields sub-pixel accuracy even for simulated high-resolution data.

Strain-induce shift of the crystal-field splitting of SrTiO3 embedded in scandate multilayers.

Strained SrTiO3 layers have become of interest, since the paraelectric-to-ferroelectric transition temperature can be increased to room temperature. A linear relationship between strain and energy splitting of the fundamental transitions in the fine structure of Ti L(2,3) and O K edges is observed, that can be exploited to measure strain from electronic transitions, complementary to measuring local strain directly via high-resolution transmission electron microscopy (HRTEM) images. In particular, for both methods, the geometrical phase analysis performed on high-resolution images and the measurement of the energy splitting by energy loss spectroscopy, tensile strain of SrTiO3 layers was measured when grown on DyScO3 and GdScO3 substrates. The effect of strain on the electron loss near edge structure (ELNES) of the Ti L(2,3) edge in comparison to unstrained samples is analyzed. Ab initio calculations of the Ti L(2,3) and O K edge show a linear variation of the crystal field splitting with strain. Calculated and experimental values of the crystal field splitting show a very good agreement.

Plastic-deformation mechanism in complex solids.

In simple crystalline materials, plastic deformation mostly takes place by the movement of dislocations. Although the underlying mechanisms in these materials are well explored, in complex metallic alloys--crystalline solids containing up to thousands of atoms per unit cell--the defects and deformation mechanisms remain essentially unknown. Owing to the large lattice parameters of these materials, extended dislocation concepts are required. We investigated a typical complex metallic alloy with 156 atoms per unit cell using atomic-resolution aberration-corrected transmission electron microscopy. We found a highly complex deformation mechanism, based on the movement of a dislocation core mediating strain and separate escort defects. On deformation, the escort defects move along with the dislocation core and locally transform the material structure for the latter. This mechanism implies the coordinated movement of hundreds of atoms per elementary glide step, and nevertheless can be described by simple rearrangement of basic structural subunits.

StripeSTEM, a technique for the isochronous acquisition of high angle annular dark-field images and monolayer resolved electron energy loss spectra.

A technique capable of producing monolayer resolved electron energy loss (EEL) spectroscopy data along one direction in crystal structures is introduced. Unambiguous assignment of EEL spectra to atomic planes is possible via the execution of high angle annular dark-field (HAADF) imaging and EEL spectrum acquisition in parallel. The recording of instrumental instabilities in the HAADF image during the measurement enables a proper quantification by virtue of post-acquisition correction. Compared to the conventional line profile technique a dose reduction by several orders of magnitude can be achieved. The technique is applied to bulk SrTiO(3) and ZnO:In(2)O(3) in order to explore its capabilities and limits. Monolayer resolution was achieved for the Ti-L(23) and In-M(45) core-losses. Multislice calculations were carried out for the purpose of assessing the residual delocalisation of the inelastic signal. Fundamental limits to the resolution are imposed by dynamical dispersion of the electron wave in the crystal combined with the extension of the inelastic potential. In the present case, owing to the requirement of a high beam current, the geometrical probe size cannot be neglected when compared to the width of an inelastic scattering potential.

Defects induced on chemical vapour deposition carbon nanotubes during peapod synthesis on substrates.

Individual carbon nanotubes are filled with fullerene molecules directly on the substrate. Two different oxidation techniques for opening the tubes prior to the filling, annealing in air, and acid treatment, are compared. High-resolution transmission electron microscopy images indicate that both methods induce defects on the sidewalls of the nanotubes. In the case of acid treatment, the inner walls can be damaged without affecting the outer walls, while the inner walls are opened along with the outer ones by heating in air. The effect of acid treatment on the tubes is much stronger than known from bulk samples. In contrast to previous studies, we find amorphous carbon inside the nanotubes after oxidation, and an additional high-temperature annealing step is needed to remove these plugs in order to open the tubes for filling.

Atomic-precision determination of the reconstruction of a 90 degree tilt boundary in YBa2Cu3O7-delta by aberration corrected HRTEM.

Aberration corrected high-resolution transmission electron microscopy is used to determine the reconstruction of atomic bonds of a 90 degree [100] grain boundary in YBa(2)Cu(3)O(7-delta). A precise measurement of atom positions within the grain boundary and the assessment of the oxygen stoichiometry require at the same time a high control of residual lens aberrations of the electron microscope and a good signal-to-noise ratio. This goal is achieved by the combination of spherical-aberration correction in the microscope with the numerical exit-plane wave function reconstruction from focal series. Atomic column positions for individual cations and anions are determined by the regression analysis of peak maxima in the phase image of the retrieved exit-plane wave function. The measurement accuracy is quantitatively assessed, including the statistical error related to residual noise. Changes in bondlengths between copper atoms and the apical oxygen are measured, indicating the distortion of the square pyramidal oxygen coordination of plane copper sites and the square coordination of chain copper sites in the grain boundary.

P-selectin and MAC-1 mediate monocyte rolling and adhesion to ECM-bound platelets under flow conditions.

Accumulation of monocyte-derived foam cells in focal areas of the atherosclerotic (A.S.-) lesion is one of the key events in early atherogenesis. Using a flow model for the damaged vessel wall, we examined the ability of ECM-bound platelets to induce monocyte tethering and adhesion. Whereas ECM-proteins alone induced monocyte adhesion only at low shear stresses (< 100 mPa), ECM-bound platelets induced monocyte rolling and adhesion at shear stresses up to 240 mPa. Studies with specific antibodies showed that monocyte adhesion to platelets was mainly mediated by P-selectin and monocyte PSGL-1 (maximum inhibition 90%). beta2-Integrin blocking CD18 and CD11b antibodies partly inhibited the arrest of rolling cells. Antibodies against other adhesion molecules such as LFA-1, PECAM-1, and beta1-integrins had no effect. Even sparsely adhered platelets (approximately 10% coverage of the surface) already strongly supported monocyte tethering. In conclusion, activated platelets present on ECM are a powerful adhesive substrate for monocyte recruitment under flow conditions.

Out-of-hospital cardiac arrest in the 1990's: a population-based study in the Maastricht area on incidence, characteristics and survival.

OBJECTIVES: We sought to describe the incidence, characteristics and survival of out-of-hospital sudden cardiac arrest (SCA) in the Maastricht area of The Netherlands. BACKGROUND: Incidence and survival rates of out-of-hospital SCA in different communities are often based on the number of victims resuscitated by the emergency medical services. Our population-based study in the Maastricht area allows information on all victims of witnessed and unwitnessed SCA occurring outside the hospital. METHODS: Incidence, patient characteristics and survival rates were determined by prospectively collecting information on all cases of SCA occurring in the age group 20 to 75 years between January 1, 1991 and December 31, 1994. Survival rates were related to the site of the event (at home vs. outside the home) and the presence or absence of a witness and rhythm at the time of the resuscitation attempt in out-of-hospital SCA. RESULTS: Five hundred fifteen patients were included (72% men, 28% women). In 44% of men and 53% of women, SCA was most likely the first manifestation of heart disease. In patients known to have had a previous myocardial infarction (MI), the mean interval between the MI and SCA was 6.5 years, with >50% having a left ventricular ejection fraction >30%. The mean yearly incidence of SCA was 1 in 1,000 inhabitants. Of all deaths in the age groups studied, 18.5% were sudden. Nearly 80% of SCAs occurred at home. In 60% of all cases of SCA a witness was present. Cardiac resuscitation, which was attempted in 51% of all subjects, resulted overall in 32 (6%) of 515 patients being discharged alive from the hospital. Survival rates for witnessed SCA were 8% (16 of 208 subjects) at home and 18% (15 of 85 subjects) outside the home (95% confidence interval 1% to 18.8%). CONCLUSIONS: The majority of victims of SCA cannot be identified before the event. Sudden cardiac arrest usually occurs at home, and the survival of those with a witnessed SCA at home was low compared with that outside the home, indicating the necessity of optimizing out-of-hospital resuscitation, especially in the at-home situation.

A clinicopathological study on overexpression of cyclin D1 and of p53 in a series of 248 patients with operable breast cancer.

Overexpression of cyclin D1 is frequently found in various types of human tumours and results from clonal rearrangement and/or amplification involving chromosomal region 11q13. In order to evaluate the pathological relevance of cyclin D1 overexpression in human breast cancer, we generated a polyclonal antiserum against the carboxy-terminal part of the cyclin D1 protein. After affinity purification, the antiserum specifically detected overexpression of cyclin D1 in formalin-fixed, paraffin-embedded tumour material also. The intensity of the nuclear stainings was, in general, proportional to the degree of cyclin D1 amplification. We did not encounter significant variability of staining within individual tumours with overexpression of cyclin D1. Overexpression of cyclin D1 appeared to be associated with oestrogen receptor-positive breast tumours, but not with any other clinicopathological parameter tested. Overexpression of cyclin D1 was not prognostic value for recurrence of survival in a consecutive series of 248 operable breast cancer patients (stage I and II). Overexpression of p53 was also not of prognostic significance in this series, but was associated with undifferentiated histology and oestrogen receptor-negative breast tumours, as has been reported previously by others. A high proportion of breast tumours with a low grade of malignancy in this series of operable breast cancer patients may explain discrepancies concerning the prognostic value of amplification and of overexpression of cyclin D1.

C5a-induced migration of human monocytes is primed by dexamethasone.

Allergic inflammation in the lung is characteristic of allergic asthma. This inflammatory process is inhibited by treatment with glucocorticoids. One of the cell types involved in the inflammatory process, the monocyte, is found in enhanced numbers in mucosal lung biopsies of asthmatic patients. Little is known about the mechanisms that lead to increased numbers of monocytes in lung tissue. We studied one of the processes involved, chemotaxis, in a modified Boyden Chamber assay. The effect of the antiinflammatory drug dexamethasone was tested on monocyte chemotactic responses to complement fragment C5a. Human monocytes from peripheral blood of normal human volunteers were purified by centrifugal elutriation. The monocytes showed a reproducible chemotactic response toward C5a with an optimum at a concentration of 10(-9) M. After culture of the monocytes overnight, the monocyte responses were clearly impaired. It is interesting that upon culture, dexamethasone increased monocyte chemotaxis in a dose-dependent manner. Analysis of the filters with an image analyzer showed that the effect was not through modulation of a subpopulation of monocytes. This steroid effect was specific and modulated via steroid receptors, because the introduction of RU 38486, a steroid receptor antagonist, completely inhibited the effect of dexamethasone. These findings are a further illustration of the complex mechanisms involved in the orchestration of the inflammatory response in asthma.

Mast cell heterogeneity in human lung tissue.

1. In this study mast cells were found to comprise 2.1% of total cells recovered by enzymatic digestion of human lung tissue. 2. This mast cell population consisted of 79% formalin-sensitive, Alcian Blue-positive mast cells and 21% formalin-insensitive, Alcian Blue-positive mast cells. 3. By the use of centrifugal elutriation and subsequent Percoll gradient centrifugation, separate mixed cell populations could be obtained in which the mast cell constituents were either of the formalin-sensitive or -insensitive type. 4. Cell suspensions in which formalin-sensitive cells comprised 97% of mast cells contained approximately 1.34 pg of histamine per mast cell, whereas in preparations in which mast cells were 84% formalin-resistant the histamine content was approximately 4.17 pg of histamine per mast cell. 5. The histamine release upon anti-immunoglobulin E challenge of formalin-sensitive mast cells was greater than the release by formalin-insensitive mast cells. 6. After challenge with opsonized zymosan, only formalin-sensitive mast cells were able to release histamine. 7. Leukotriene C4 release was observed when formalin-sensitive mast cells were challenged with anti-immunoglobulin E. Formalin-insensitive mast cells showed no release of leukotriene C4. 8. Prostaglandin D2 release was observed when formalin-insensitive mast cells were challenged with anti-immunoglobulin E. Formalin-sensitive mast cells showed no release of prostaglandin D2.

Directional cellular movement of cell populations: its description by chemotactic assays.

Chemotaxis (CT) of cells is an important factor in the defense of organisms. For the study of this process several assays are available using the Boyden chamber. Its popularity, however, has led to a use of the system of which the basic qualities are not always known and so may lead to erroneous interpretation of results. Experiments were performed with the purpose of elucidating the significance of parameters regularly used in CT: 1. the cell count at the bottom of the permeable filter, 2. a chemotactic index, and 3. the leading front method. Neutrophils and eosinophils were stimulated with FMLP (10(-13)-10(-3) M). It appeared that: 1. the parameters are indicative only for (small) parts of the cell population, 2. for the comparison of effects of compounds complete concentration-effect relationships are needed, 3. to avoid cell to cell interactions a maximal cell purity is desired, and 4. if possible, no combinations of chemotactic agents should be tested.

Patients with chronic bronchitis differ in their mast cell subtypes as compared with normal subjects.

Mast cells were isolated by enzymatic digestion from lung tissue obtained from patients with chronic obstructive lung disease and from normal subjects. Two mast cell subtypes could be demonstrated in human lung tissue. Mast cell subtypes were differentiated in formalin-sensitive and formalin-insensitive mast cells. It appeared that compared with normal individuals, patients suffering from chronic bronchitis had increased numbers of mast cells of the formalin-sensitive type, whereas patients with emphysema had reduced numbers, but the same ratio, of both mast cell subtypes.

Mast cell subtypes from human lung tissue: their identification, separation, and functional characteristics.

The contribution of mast cell subtypes and their different mediators to the pathogenesis of chronic obstructive lung diseases (COLD) has not yet been established. In the present study, enzymatic digestion, centrifugal elutriation and Percoll gradient centrifugation were used to obtain two populations of mast cell subtypes from human lung tissue. Mast cell subtypes were challenged with anti-human IgE, propranolol, compound 48/80, or opsonized zymosan. Both subtypes were able to release histamine, but differed in the amount of the amine release. Only the formalin-sensitive and alcian blue-positive type (FS-AB) released histamine on challenge with opsonized zymosan. The same subtype was able to release leukotriene C4 (LTC4) after challenge with anti-human IgE. The other subtype, the formalin-insensitive and alcian blue-positive type (FI-AB), did not respond to opsonized zymosan and did not release LTC4 after challenge with anti-human IgE. Stimulation with propranolol or compound 48/80 did not release histamine from the FS-AB mast cells while the FI-AB mast cells released only about 10% of their histamine content upon challenge with these secretagogues.