Improved accuracy in DFS pattern interpretation using a novel HEp-2 ELITE system.

INTRODUCTION/OBJECTIVES: Accurate interpretation of DFS70 (dense fine speckled 70) and mixed antinuclear antibodies (ANAs) patterns can be challenging using conventional HEp-2 immunofluorescence (IIF) method. We evaluated a novel HEp-2 IIF substrate (HEp-2 ELITE/DFS70-KO) composed of a mixture of engineered HEp-2 devoid of the DFS70 autoantigen and conventional HEp-2 cells. The study assessed the utility of the new substrate in ANA screening and its advantages. METHOD: One thousand and five consecutive routine samples sent for ANA screening were tested on both standard HEp-2 and the HEp-2 ELITE DFS70 KO substrates (ImmuGlo ANA HEp-2 and HEp-2 ELITE/DFS70-KO, Trinity Biotech, Buffalo, NY). Anti-DFS70 antibody specificity was additionally determined by immunoblot (IB). Clinical and serological data were included in the analysis of the overall impact of the novel HEp-2 substrate on DFS pattern interpretation. RESULTS: Of the 22 cases suspected as positive for DFS pattern alone or in combination with homogeneous or speckled patterns on conventional HEp-2 cells, 17 were interpreted with a higher accuracy using the new HEp-2 ELITE method as positive for DFS70 (monospecific DFS70 (10), mixed DFS70 (7)), speckled (3), and DFS (2) patterns. CONCLUSIONS: The new substrate was not only useful in deciphering unclear mixed ANA patterns but also highly sensitive in detecting DFS70 pattern in comparison to the DFS70 positivity obtained using IB.

Reflection and transmission of guided ultrasonic plate waves by vertical stiffeners.

Ultrasonic guided waves are very useful for structural health monitoring. They have the potential to interrogate and detect damage in a structure over a large area with few transducers. Guided plate modes (Lamb waves) are used and proposed for damage detection at a distance in semi-monococque structures such as airplane fuselages and spacecraft structures. The interaction of guided plate waves with stiffening members such as ribs, stringers, or the integral stiffeners used in spacecraft structures limits the distance over which structural health monitoring non-destructive evaluation systems can detect damage. This paper develops a simple explanatory model for the scattering of low-order ultrasonic Lamb waves crossing a stiffening device. The model illuminates the underlying mechanics of waves crossing a stiffener. The model shows that stop-bands for transmission of S(0) (longitudinal pressure) waves across a stiffener line up with flexural resonances of the stiffener. It also demonstrates why transmission of A(0) (flexural) waves is more complicated and harder to predict. The model is shown to agree well with both boundary element method calculations and experimental measurements.

Review of air-coupled ultrasonic materials characterization.

This article presents a review of air-coupled ultrasonics employed in the characterization or nondestructive inspection of industrial materials. Developments in air-coupled transduction and electronics are briefly treated, although the emphasis here is on methods of characterization and inspection, and in overcoming limitations inherent in the use of such a tenuous sound coupling medium as air. The role of Lamb waves in plate characterization is covered, including the use of air-coupled acoustic beams to measure the elastic and/or viscoelastic properties of a material. Air-coupled acoustic detection, when other methods are employed to generate high-amplitude sound beams is also reviewed. Applications to civil engineering, acoustic tomography, and the characterization of both paper and wood are dealt with here. A brief summary of developments in air-coupled acoustic arrays and the application of air-coupled methods in nonlinear ultrasonics complete the review. In particular, the work of Professor Bernard Hosten and his collaborators at Bordeaux is carefully examined.

Cell surface expression of activating receptors and co-receptors on peripheral blood NK cells in systemic autoimmune diseases.

OBJECTIVES: A defined role for natural killer (NK) cells and their activating receptors in autoimmunity has not been clearly established. The aim of this study was to evaluate the levels of the CD3-CD56+ NK cells and their expression of receptors and co-receptors in the peripheral blood of patients with systemic autoimmune disorders. METHODS: Thirty-four subjects with systemic sclerosis (SSc), 14 with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), 14 with systemic lupus erythematosus (SLE), and 14 healthy donors were studied. The activating receptors NKp46, NKp44, NKp30, NKG2D, and DNAM-1 and the co-receptors NTB-A and 2B4 were analysed by flow cytometry on peripheral blood NK cells. RESULTS: In SSc, AAV, and SLE we detected a significant decrease in the percentage of CD3-CD56+ NK cells compared to healthy controls. No differences in the expression of NKp46, NKp44, and NKp30 were identified. On the contrary, NKG2D and DNAM-1 expression was decreased in SLE, but not in SSc and AAV, NTB-A was decreased in SLE, and 2B4 in both SLE and SSc. No differences were detected between active and inactive SLE patients. In SSc, only patients affected by pulmonary arterial hypertension (PAH) and interstitial lung disease (ILD) had a low expression of DNAM-1, 2B4, and NKp30. CONCLUSIONS: These data demonstrate that patients with different systemic autoimmune diseases differ in the expression of activating receptors and co-receptors on CD3-CD56+ NK cells. The down-regulation of receptors and co-receptors in SSc with lung involvement suggests their possible role in this manifestation of the disease.

Guided plate wave scattering at vertical stiffeners and its effect on source location.

This paper examines guided wave transmission characteristics of plate stiffeners and their influence on the performance of acoustic noise source location. The motivation for this work is the detection of air leaks in manned spacecraft. The leaking air is turbulent and generates noise that can be detected by a contact-coupled acoustic array to perform source location and find the air leak. Transmission characteristics of individual integral stiffeners are measured across a frequency range of 50-400kHz for both high and low aspect-ratio rectangular stiffeners, and comparisons are made to model predictions which display generally good agreement. It is demonstrated that operating in frequency ranges of high plate wave stiffener transmission significantly improves the reliability of noise source location in the plate. A protocol is presented to enable the selection of an optimal frequency range for leak location.

Role of endothelial progenitor cell mobilization after percutaneous angioplasty procedure.

Circulating endothelial progenitor cells (EPCs) are bone marrow-derived cells, contributing to endothelial cell regeneration of injured vessels as well as neovascularization of ischemic lesions. EPC levels and function are inversely correlated with cardiovascular risk factors, can predict the occurrence of adverse events and atherosclerotic disease progression. Ischemia and inflammation are the primary triggers for EPC mobilization and homing, however, vascular trauma, as it occurs during surgical procedures, has been demonstrated to stimulate EPC mobilization even in absence of tissue ischemia. The effect of angioplasty on EPCs is not well defined, mainly because of the different and sometimes contrasting clinical results, due to low numbers of patients enrolled and to lack of standardization in evaluating EPCs. Aim of this review is to report recent results on the effect of EPC mobilization and homing after angioplasty, attempting to summarize them in a comprehensive model. The effect on EPCs of different kind of stents and the potential use of new stents able to attract EPCs will be also described. Results obtained in patients undergoing angioplasty in different vascular districts (coronary, peripheral and carotid) will be shown, together with the correlation between circulating progenitor cells and restenosis.

Effects of different LDL particles on inflammatory molecules in human mesangial cells.

AIMS/HYPOTHESIS: Inflammation is a mechanism of glomerular damage in chronic glomerulopathies. LDL may increase the production of inflammatory cytokines in renal tissues. However, the relative role of native, oxidised and glycated LDL in promoting this process has been only partially elucidated. METHODS: We tested the inflammatory and proapoptotic effects of native, oxidised and glycated LDL in human mesangial cells (HMCs) by measuring levels of IL6, CD40 and macrophage migration inhibitory factor (MIF) genes, MIF protein, release of IL6, soluble CD40, fibronectin and laminin, early and late apoptosis, and extracellular regulated kinases (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation. RESULTS: IL6 and CD40 mRNA were dose-dependently upregulated by all three species; this was closely paralleled by their increased release. MIF mRNA was potently stimulated by modified LDL, as confirmed by immunostaining. Fibronectin and laminin release was stimulated by both oxidised and glycated, but not native, LDL. All LDL species induced some increase in late, but not early, apoptosis, and similarly activated JNK2/3 phosphorylation; in contrast, ERK1/2 phosphorylation was more strongly upregulated by oxidised than either native or glycated LDL. CONCLUSIONS: In HMCs, the production and release of IL6 and CD40 is stimulated by both native and modified LDL, while MIF is more strongly stimulated by oxidised LDL. Regarding the pattern of mesangial expansion, fibronectin and laminin are upregulated by oxidised and glycated LDL. Apoptosis, if modest, is induced by all species. Intracellular signalling of native and modified LDL involves JNK2/3 and, perhaps more specifically, ERK1/2. Tight control of the lipid profile may be useful in preserving kidney function in patients with metabolic alterations.

IgG, IgA, IgM antibodies to a viral citrullinated peptide in patients affected by rheumatoid arthritis, chronic arthritides and connective tissue disorders.

OBJECTIVES: Anti-citrullinated protein/peptide antibodies (ACPA), a family of antibodies with overlapping specificities, represent a specific marker of rheumatoid arthritis (RA). The aim of the present study is to investigate the prevalence and clinical significance of IgG, IgA and IgM ACPA by a newly described assay employing a viral citrullinated peptide (VCP). METHODS: IgG, IgA and IgM anti-VCP antibodies have been measured in sera from 146 patients affected by RA and 404 controls, including 204 chronic arthritides, 111 connective tissue disorders and 89 healthy subjects. The affinity of the different isotypes for VCP was analysed by liquid phase inhibition assays. RESULTS: Among RA patients, 40 were single positive for IgG anti-VCP, five for IgA and 11 for IgM. Ten patients were double positive for IgG and IgA, four for IgG and IgM, six for IgA and IgM. In 15 RA patients IgG, IgA and IgM anti-VCP antibodies were detected. No correlation could be found between the isotype and the clinical manifestations or duration of the disease. IgA anti-VCP were strongly associated with RA, whereas IgM anti-VCP were detected also in a low percentage of systemic lupus erythematosus, psoriatic arthritis and mixed cryoglobulinaemia (MC) patients. IgG anti-VCP displayed a higher affinity for the antigen than IgA or IgM. CONCLUSIONS: These data show that anti-VCP of IgG and IgA isotype discriminate RA from other chronic arthritides and disease controls and suggest an independent production of each isotype.

Anti-C1q antibodies in pregnant patients with systemic lupus erythematosus.

OBJECTIVE: To study anti-C1q antibodies in pregnant patients with systemic lupus erythematosus (SLE) and to evaluate their prognostic significance for the occurrence of disease flares or pregnancy complications. METHODS: Twenty-one pregnancies in 19 SLE patients prospectively followed were analyzed. Disease activity was evaluated on the basis of the physician's intention to treat and a modified version of the ECLAM index. Anti-C1q and anti-dsDNA antibodies were detected in the sera by an ELISA assay. Antinuclear antibodies, anti-ENA antibodies, anticardiolipin antibodies and lupus anticoagulant were also performed. RESULTS: In all the patients the disease was inactive at the beginning of the pregnancy. Four flares of disease activity were observed in 4 pregnancies (19%) and obstetric complications were encountered in 7 pregnancies (43%). Anti-C1q antibodies were positive in 4 (19%) pregnancies and anti-dsDNA antibodies in 8 (38%). The presence of anti-phospholipid antibodies at the first assessment was correlated with the occurrence of obstetric complications (p<0.05). The presence of anti-C1q and anti-dsDNA antibodies at the first assessment had no prognostic significance for the occurrence of flares or obstetric complications during the course of pregnancy. Although the small number of patients studied did not allow for statistically significant analysis, flares appeared to be more likely to occur in patients presenting with anti-dsDNA or anti-C1q antibodies during pregnancy compared to patients with no changes in these antibody titers (43% vs 8% respectively). CONCLUSIONS: The presence of anti-C1q and anti-dsDNA antibodies does not seem to be prognostic for the occurrence of flares during pregnancy. Further studies are warranted to explore this possibility.

Locating air leaks in manned spacecraft using structure-borne noise.

All manned spacecraft are vulnerable to leaks generated by micrometeorite or debris impacts. Methods for locating such leaks using leak-generated, structure-borne ultrasonic noise are discussed and demonstrated. Cross-correlations of ultrasonic noise waveforms from a leak into vacuum are used to find the location of the leak. Four methods for sensing and processing leak noise have been developed and tested and each of these can be used to reveal the leak location. The methods, based on phased-array, distributed sensor, and dual sensor approaches, utilize the propagation patterns of guided ultrasonic Lamb waves in the spacecraft skin structure to find the source or direction of the leak noise. It is shown that each method can be used to successfully locate the leak to within a few millimeters on a 0.6-m2 aluminum plate. The relative merits of the four methods are discussed.

An ultrasonic array sensor for spacecraft leak direction finding.

We have developed an ultrasonic array sensor useable for locating air leaks in manned spacecraft and have found that this sensor locates leaks in a 1-m(2) plate to within 2 cm. The sensor consists of a 63-element multiplexed array plus a reference element, all constructed from a single PZT disc and a printed circuit board. Cross-correlations of signals from the array elements with signals from the single reference element provide a measurement of the leak noise passing through the spacecraft skin under the array. A spatial Fourier transform reveals the dominant direction of propagation. Triangulation from multiple sensor locations can be used to find the source of the leak.

High contrast air-coupled acoustic imaging with zero group velocity lamb modes.

The well known zero in the group velocity of the first-order symmetric (S1) plate wave mode has been exploited in air-coupled ultrasonic imaging to obtain significantly higher sensitivity than can be achieved in conventional air-coupled scanning. At the zero group velocity point at the frequency minimum of the S1 mode, a broad range of wavenumbers couple into the first-order symmetric mode at nearly a constant frequency, greatly enhancing transmission at that frequency. Coupled energy remains localized near the coupling point because the group velocity is zero. We excite the mode with a broadband, focussing, air-coupled transducer at the frequency of the zero group velocity point in the S1 mode. By exploiting the efficient coupling at the zero group velocity frequency, we have easily imaged a single layer of Scotch tape attached to a 6.4-mm thick Plexiglas plate and 3.2-mm Teflon inserts in a composite laminate.

Material property estimation in thin plates using focused, synthetic-aperture acoustic beams.

The method developed here exploits the wide angular range of focused acoustic probes and the large synthetic aperture of scanned transducers to permit a rapid and reliable estimation of material properties in thin plates. It is found in several tests with various materials that estimates of elastic behavior using this method agree with contact measurements to within less than 5%. The method utilizes transmission (or reflection) coefficient reconstruction for an infinite thin plate, across a wide range of frequency and wave number, from which elastic property estimates are made. Data collected over a large synthetic acoustic aperture are processed with temporal and spatial Fourier transforms applied to change the acquired data from the coordinate and time domains to the wave number and frequency domains. Extrinsic real-beam effects on the data are accounted for with a complex transducer point analysis. Transmission measurements yield reconstructed data extending to the mode cutoffs, permitting easy and nearly unambiguous estimation of a subset of the elastic stiffnesses. For anisotropic plates, elastic stiffnesses are estimated with an inversion procedure that uses only limited data carefully selected from different portions of the measured scattering coefficient. Estimates are made by reconstructing in a stepwise fashion, based on sensitivity studies, where only one stiffness is estimated from the data at any one time, restricting the optimization to a robust one-dimensional search.

Microwave excitation of ultrasound in graphite-fiber reinforced composite plates.

In this paper is demonstrated the effect of microwave beam polarization on the thermal generation of acoustic waves in continuous fiber-reinforced composite laminates. It is found that beam polarization strongly influences the dielectric interaction that leads to thermal losses, bulk expansion, and acoustic wave generation. The oriented graphite fibers in the composite laminate effectively short the microwave fields and reduce the generation efficiency nearly to zero. Ultrasonic waves at several hundred kHz generated in the composite are detected by air-coupled acoustic transducers located on the opposite side of the plate specimen from the 9.41 GHz incident microwave beam. With some averaging signal-to-noise ratios of better than 26 dB are obtained. Applying a conventional model of electromagnetic wave scattering in anisotropic media to this experiment yields good agreement between calculations and measured data. Implications for microwave-acoustic testing of graphite-reinforced composites are also discussed.

Equivalence of Gaussian and piston ultrasonic transducer voltages.

A plane-wave decomposition of collimated beams and electromechanical reciprocity relations are used to demonstrate fundamental differences and unusual similarities about transducer fields and transducer voltages at ultrasonic frequencies under various conditions. It is shown that the voltage induced by a transmitting acoustic piston transducer (constant particle velocity over the transducer surface) radiating into an ideal fluid medium on a second identical piston transducer, operating as a receiver, is nearly identical to the voltage observed when the two transducers have instead a Gaussian radial surface velocity distribution. The strong similarity in induced voltage begins when the two devices are separated by only several acoustic wavelengths, still well within the nearfield of both transducers, and the similarity increases with separation. Cases of transducer velocity distributions different from piston, such as Lorentzian or triangular, yield results nearly identical to the piston case. Transducers of differing size are also treated. It is further shown that an "equivalent transducer" can be derived for any combination of radiators, whose field is identical to the voltage measured using the pair. Moreover, with this concept the calculation of voltage in two-probe experiments is as simple as well-known approximations to the Rayleigh integral for a single transducer. These results have substantial consequences for calculations, either analytical or numerical, that predict the voltage measured in two-transducer experiments.

Anti-ribosomal antibodies from lupus patients bind DNA.

Antibodies specific for ribosomal P proteins (anti-P) are a hallmark of systemic lupus as anti-DNA antibodies are. It has been reported that anti-P antibodies are more frequently detected in anti-dsDNA positive sera. The aim of the present study was to verify the binding ability of anti-P antibodies towards polynucleotides and nucleosomes. We purified anti-P antibodies from 8 SLE patients' sera and we analysed them by ELISA on plates coated with DNA or nucleosomes. We performed also inhibition experiments in order to verify the specificity of the binding. All the purified anti-P antibodies bound DNA, but some anti-DNA binding activity remained among the non-anti-P antibodies in the flow through. Only half of the purified antibodies bound to nucleosomes, and anti-nucleosomal activity was demonstrated also in non anti-P antibody fraction. The inhibition experiments performed on two anti-P antibodies pointed out that only the homologous antigen inhibited the binding to either P peptide or DNA coated onto the solid phase. These results show that sera in which the two specificities coexist contain antibodies endowed with a double binding ability for DNA and the P peptide.

Acoustic waves generated by pulsed microwaves in viscoelastic rods: modeling and experimental verification.

The acoustic wave generation in a specimen irradiated by a pulsed microwave is predicted theoretically. The specimen is a viscoelastic rod inserted into a wave guide. The model is based on Maxwell's equations, heat equation and thermoviscoelasticity theory. Computations show the presence of temperature oscillations due to the electromagnetic interferences in the irradiated rod if its electromagnetic absorption is low. An experimental method to infer indirectly the detailed behavior of microwave-generated acoustic waves in polymer rods, including the influence of electromagnetic wave reflection at the rod ends, is presented. The method consists of measuring the oscillations in the particle acceleration detected at the end of the rod that are induced by variations in the polymer rod length. The oscillations are caused by changing electromagnetic standing-wave conditions within the rod. It is found that these oscillations are in agreement in period, amplitude, and phase, with independent values of the complex dielectric constant and complex acoustic slowness of the polyvinyl chloride samples used in the study.

European genetic study on rheumatoid arthritis: is there a linkage of the interleukin-1 (IL-1), IL-10 or IL-4 genes to RA?

UNLABELLED: The genetic predisposition for rheumatoid arthritis (RA) is only partly explained by the HLA locus and most genetic factors involved in the susceptibility (and/or severity) of the disease await further identification. The first European genome scan in RA families provided suggestive evidence for linkage with a region (3.1/3q13) on chromosome 3, but many other potential RA susceptibility genes have yet to be analysed. AIMS: To perform a linkage analysis with microsatellite markers located in the vicinity of the interleukin-1 (IL-1) gene superfamily, the IL-10 gene and the IL-4 gene cluster which might be considered putative candidate loci for RA. METHODS: 107 Caucasoid European RA sibpairs from 90 nuclear families were genotyped for markers flanking the genes for the IL-1 superfamily, IL-10 and the IL-4 gene cluster. Linkage analysis based on the identity by descent (IBD) in affected siblings was analysed with the program SIBPALNA. Affected sibpairs were stratified according to the identity by state (IBS) for three markers in the HLA region (DRB1 oligotyping, D6S276 and TNFa microsatellites) and to the presence/absence of erosive disease on X-ray examination. RESULTS: Analysis of the whole family set showed an excess of allele sharing for markers of the IL-1 gene cluster (IBD 60%; P = 0.012) but not for IL-10 or IL-4. After stratification, the evidence of linkage to IL-1 was restricted to HLA concordant sibpairs (n = 32; IBD 70%; P = 0.006). Some evidence of linkage to IL-10 was also observed in HLA concordant sibpairs (IBD 66%; P = 0.03) and in sibpairs with erosive disease (n = 61; IBD 62%; P = 0.02). CONCLUSIONS: We found suggestive evidence of linkage of RA to the IL-1 locus. The increased linkage to IL-1 and IL-10 in HLA-identical sibs suggests a possible interaction between these cytokines and the HLA loci. Moreover IL-10 could interact with HLA factors in predisposing to erosive disease. These results need to be tested in additional families for consistency and replication.

Induction of anti-DNA antibodies in preautoimmune NZBxNZW F1 mice by immunization with a DNA-DNase I complex.

Recent studies suggest that anti-DNA antibodies may arise from the immune response to a complex of DNA and a DNA-binding protein. One of the protein targets frequently recognized by anti-DNA antibodies is the enzyme DNase I. To investigate the possible role of DNase I in the induction of anti-DNA antibodies, we immunized preautoimmune NZBxNZW F1 mice with a complex of DNA and DNase I emulsified in complete Freund's adjuvant. Control mice received DNA or DNase in adjuvant. IgG anti-dsDNA antibodies were induced in 50% of the mice immunized with DNA-DNase, in 25% of the mice immunized with DNase and in 6% of the mice immunized with DNA. However, immunized mice that produced anti-DNA antibodies did not develop renal disease. These data show that a DNA-binding protein like DNase may act as carrier in the immune response that leads to anti-DNA antibodies production in an autoimmune strain, but the induced anti-dsDNA antibodies have a low pathogenic potential.

Autoantibodies specific for alpha-enolase in systemic autoimmune disorders.

OBJECTIVE: To analyze the presence and specificity of anti-alpha-enolase antibodies in various systemic autoimmune diseases. METHODS: Sera from patients with systemic lupus erythematosus (SLE), mixed cryoglobulinemia (MC), systemic sclerosis (SSc), and rheumatoid arthritis (RA) were tested by immunoblot on partially purified a-enolase from human kidney and on beta- and gamma-enolase. The isotype of anti-enolase antibodies was determined by means of isotype-specific monoclonal antibodies. RESULTS: IgG anti-alpha-enolase antibodies were detected in 9/33 (27%) SLE sera (6/9 patients had active renal disease), in 6/19 sera from patients with MC and nephritis, in 0/15 sera from MC patients without renal involvement, in 6/20 (30%) SSc sera, in 2/35 (6%) disease controls with RA, and in 2/32 (6%) healthy controls. The antibodies were not species-specific, but in most cases were specific for the alpha isoform of enolase. The anti-enolase immune response was not isotypically restricted. In half of the patients with SLE the anti-alpha-enolase and anti-DNA antibodies constituted distinct antibody populations, while in the other half a partial overlap of the 2 antibody specificities was observed. CONCLUSION: Anti-alpha-enolase antibodies can frequently be detected in systemic autoimmune disorders. In SLE and MC they are associated with nephritis and in SSc they are associated with severe endothelial damage. Alpha-enolase is ubiquitous, but is highly expressed in the kidney and also on the membrane of several cell types including endothelial cells. Thus, anti-alpha-enolase antibodies could contribute to renal injury not only by the local formation of immune complexes, but also by direct damage to endothelial cells.