Increased B-cell activating factor, interleukin-6, and interleukin-8 in induced sputum from primary Sjögren's syndrome patients.

OBJECTIVE: Small airway disease and chronic obstructive pulmonary disease are common in primary Sjögren's syndrome (pSS). However, the underlying inflammatory mechanisms behind pSS-associated airway disease have not been studied in detail. We therefore wanted to study cytokine and leucocyte levels in induced sputum in never-smoking patients with pSS. METHOD: Induced sputum cytokines and leucocytes were assessed in 20 never-smoking patients with pSS and 19 age- and gender-matched population-based controls. In addition, pulmonary function, disease activity, respiratory symptoms, and inflammatory and serological features of pSS were assessed. RESULTS: B-cell activating factor (BAFF), interleukin-6 (IL-6) and IL-8 were significantly increased in induced sputum in pSS patients compared to population-based controls, while IL-1ß, interferon-α, and tumour necrosis factor-α levels and leucocytes were not. The proportion of lymphocytes and BAFF levels in induced sputum correlated significantly in pSS patients. However, cytokine levels in induced sputum were not associated with pulmonary function tests, disease activity, respiratory symptoms, or serological features of pSS. CONCLUSION: The increase in BAFF, IL-6, and IL-8 in induced sputum suggests a specific ongoing inflammatory disease process in the airways in pSS patients. Its association with pSS-associated airway disease needs to be further examined in future larger studies.

Mast cell-dependent IL-33/ST2 signaling is protective against the development of airway hyperresponsiveness in a house dust mite mouse model of asthma.

Interleukin-33 (IL-33) and its receptor ST2 have been influentially associated with the pathophysiology of asthma. Due to the divergent roles of IL-33 in regulating mast cell functions, there is a need to further characterize IL-33/ST2-dependent mast cell responses and their significance in the context of asthma. This study aimed to investigate how IL-33/ST2-dependent mast cell responses contribute to the development of airway hyperresponsiveness (AHR) and airway inflammation in a mouse model of house dust mite (HDM)-induced asthma. Mast cell-deficient C57BL/6-KitW-sh (Wsh) mice engrafted with either wild-type (Wsh + MC-WT) or ST2-deficient bone marrow-derived mast cells (Wsh + MC-ST2KO) were exposed to HDM delivered intranasally. An exacerbated development of AHR in response to HDM was seen in Wsh + MC-ST2KO compared with Wsh + MC-WT mice. The contribution of this IL-33/ST2-dependent mast cell response to AHR seems to reside within the smaller airways in the peripheral parts of the lung, as suggested by the isolated yet marked effect on tissue resistance. Considering the absence of a parallel increase in cellular inflammation in bronchoalveolar lavage fluid (BALF) and lung, the aggravated AHR in Wsh + MC-ST2KO mice seems to be independent of cellular inflammation. We observed an association between the elevated AHR and reduced PGE2 levels in BALF. Due to the protective properties of PGE2 in airway responses, it is conceivable that IL-33/ST2-dependent mast cell induction of PGE2 could be responsible for the dampening effect on AHR. In conclusion, we reveal that IL-33/ST2-dependent mast cell responses can have a protective, rather than causative role, in the development of AHR.

The forced oscillation technique is a sensitive method for detecting obstructive airway disease in patients with primary Sjögren's syndrome.

OBJECTIVES: To study signs of obstructive airway disease (OAD) in patients with primary Sjögren's syndrome (pSS) using the forced oscillation technique (FOT). METHOD: Thirty-seven female pSS patients (median age 64, range 38-77 years) without previous physician-diagnosed OAD, participating in a longitudinal follow-up study of pulmonary function, and 74 female population-based controls (median age 64, range 47-77 years), also without physician-diagnosed OAD, and matched with regard to age, height, weight, and tobacco consumption, were included in the study. The pSS patients and controls were studied by the FOT, evaluating resistance and reactance of the respiratory system. RESULTS: pSS patients had significantly increased resistances at 5-25 Hz, decreased reactance at 10-35 Hz, and an increased resonant frequency (Fres) in comparison with controls. Resistance was correlated negatively and reactance positively to the vital capacity (VC), the forced expiratory volume in 1 s (FEV1), and the diffusing capacity for carbon monoxide (DLCO). Compared with controls, pSS patients with (n = 14) and without OAD (n = 21), as determined by spirometry, had significantly increased resistances at 5-25 Hz and decreased reactances at 10-35 Hz. In never-smoking subjects, identical FOT signs were found. CONCLUSIONS: pSS patients showed FOT signs of obstruction affecting both peripheral and central airways. pSS patients without spirometric signs of OAD and never-smoking pSS patients also showed clear FOT signs of obstruction. FOT therefore seems to be a sensitive method for detecting obstruction in pSS patients.

Integrated acoustic immunoaffinity-capture (IAI) platform for detection of PSA from whole blood samples.

On-chip detection of low abundant protein biomarkers is of interest to enable point-of-care diagnostics. Using a simple form of integration, we have realized an integrated microfluidic platform for the detection of prostate specific antigen (PSA), directly in anti-coagulated whole blood. We combine acoustophoresis-based separation of plasma from undiluted whole blood with a miniaturized immunoassay system in a polymer manifold, demonstrating improved assay speed on our Integrated Acoustic Immunoaffinity-capture (IAI) platform. The IAI platform separates plasma from undiluted whole blood by means of acoustophoresis and provides cell free plasma of clinical quality at a rate of 10 uL/min for an online immunoaffinity-capture of PSA on a porous silicon antibody microarray. The whole blood input (hematocrit 38-40%) rate was 50 µl min(-1) giving a plasma volume fraction yield of ≈33%. PSA was immunoaffinity-captured directly from spiked female whole blood samples at clinically significant levels of 1.7-100 ng ml(-1) within 15 min and was subsequently detected via fluorescence readout, showing a linear response over the entire range with a coefficient of variation of 13%.

Factors influencing factor VIII activity in frozen plasma.

BACKGROUNDS AND OBJECTIVES: Fresh frozen human plasma is an important raw material in the production of coagulation factor concentrates used in patients with haemorrhagic disorders. The aim of the study was to determine how the handling of plasma influences the recovery of coagulation factor VIII activity (FVIII:C), i.e. the influence of time between donation and freezing, of the freezing time and of the ice front velocity. We also studied a tentative eutectic point in human plasma. MATERIALS AND METHODS: Aliquots of plasma from 12 different donors were kept at room temperature for 2, 4 and 6 h before start of freezing. We achieved fast freezing with a freezer that blows cooled air at a high velocity on the plasma containers. Freezing times were 0.5, 1, 4 and 24 h. Temperature was registered continuously during freezing. Plasma and NaCl solutions were frozen slowly to investigate the eutectic point. RESULTS: Storage at room temperature for 6 h caused a small but statistically significant decrease in FVIII:C. Slow freezing with programmed freezing times of 4 and 24 h caused a more pronounced drop in FVIII:C as compared to that of 30 and 60 min. We found no eutectic point in plasma or in plasma with addition of 2 % (w/v) NaCl. CONCLUSION: For an optimal yield of FVIII, freezing should start within 4 h after plasma donation. We propose the use of the term 'ice front velocity' instead of 'freezing speed', taking into consideration that the volume and shape of plasma containers may differ. We found only a marginal loss of FVIII:C when the ice front velocity was 26 mm/h or faster, but a significant loss when it was 9 mm/h or slower. We recommend freezing times of 60 min or shorter. We were not able to demonstrate any eutectic point in human plasma. We therefore recommend that the term eutectic point should not be used as a reference temperature in guidelines on plasma handling.

Mechanism of the antigen formation of carvone and related alpha, beta-unsaturated ketones.

In the present study, the mechanism for the antigen formation of alpha, beta-unsaturated ketones was investigated. A series of analogues of carvone ((5R)-5-isopropenyl-2-methyl-2-cyclohexenone) with altered chemical reactivity and with retained overall structure or with retained reactivity and altered three-dimensional structure were synthesized. These analogues were tested for cross-reactivity in carvone-sensitized animals. Cross-reactivity was observed for analogue 3 ((5R)-5-isopropyl-2-methyl-2-cyclohexen-1-one). No cross-reactions were observed for analogues 1 ((2R,5R)-5-isopropenyl-2-methyl cyclohexanone) and 4 ((5R)-2,3-dimethyl-5-isopropenyl-2-cyclohexene-1-one). Both those compounds also failed to induce sensitization. These findings demonstrate that alpha, beta-unsaturated ketones form antigens after a nucleophilic attack at the beta-carbon with soft nucleophiles such as thiol in cysteine and not with the formation of a Schiff's base after a nucleophilic attack at the carbonyl carbon with nitrogen nucleophiles. Furthermore, no cross-reactivity was observed between R- and S-carvone indicating the importance of the 3-dimensional structure of haptens (and antigens) in T-cell recognition. The analogues were also tested for cross-reactivity on patients allergic to carvone. The results from the animal study were confirmed.

Structural analogues inhibit the sensitizing capacity of carvone.

The aim of the study was to investigate the effect of non-allergenic structural analogues on the sensitizing potential of carvone, a fragrance allergen. The possibility that one molecule might inhibit the allergenic activity of another molecule has been debated for 25 years. The Research Institute for Fragrance Materials states that the sensitizing activity from certain fragrance aldehydes is "quenched" by the addition of other specific chemicals. However, other studies do not confirm the results, although several attempts have been made. We used a guinea pig method designed to study the sensitizing capacity of fragrance allergens. Induction was performed with either carvone alone or with a mixture of carvone and one of two analogues. A significant difference in the response rates (p < 0.001) was observed between the animals induced with carvone alone and those induced with any of the mixtures. Our investigation shows that by using selected molecules it is possible to significantly reduce the sensitizing effect of a fragrance allergen.

MRI thermometry in phantoms by use of the proton resonance frequency shift method: application to interstitial laser thermotherapy.

In this work the temperature dependence of the proton resonance frequency was assessed in agarose gel with a high melting temperature (95 degrees C) and in porcine liver in vitro at temperatures relevant to thermotherapy (25-80 degrees C). Furthermore, an optically tissue-like agarose gel phantom was developed and evaluated for use in MRI. The phantom was used to visualize temperature distributions from a diffusing laser fibre by means of the proton resonance frequency shift method. An approximately linear relationship (0.0085 ppm degrees C(-1)) between proton resonance frequency shift and temperature change was found for agarose gel, whereas deviations from a linear relationship were observed for porcine liver. The optically tissue-like agarose gel allowed reliable MRI temperature monitoring, and the MR relaxation times (T1 and T2) and the optical properties were found to be independently alterable. Temperature distributions around a diffusing laser fibre, during irradiation and subsequent cooling, were assessed with high spatial resolution (voxel size = 4.3 mm3) and with random uncertainties ranging from 0.3 degrees C to 1.4 degrees C (1 SD) with a 40 s scan time.

Optical absorption of blood depends on temperature during a 0.5 ms laser pulse at 586 nm.

Optical properties are important parameters in port wine stain laser treatment models. In this study we investigated whether changes in blood optical properties occur during a 0.5 ms laser pulse. Blood from three volunteers was irradiated in vitro with laser pulses (radiant exposure 2-12 J cm-2, wavelength 586 nm, pulse length 0.5 ms). Reflection and transmission coefficients, measured using double integrating spheres, decreased slightly during the first part of the pulse. At 2.9 J cm-2 radiant exposure, the reflectance increased, independent of total radiant exposure of the pulse. This was caused by blood coagulation. A second sudden increase in reflection and a significant increase in transmission occurred near 6.3 J cm-2 and was accompanied by a "popping" sound, indicating rapid expansion of bubbles due to blood vaporization. A multilayered model of blood was used to fit calculated transmission coefficient curves to the measurements and determine temperature-dependent optical blood absorption. Heat diffusion was shown to be of minor importance. A 2.5-fold increase in absorption for temperatures increasing from 20 to 100 degrees C, accurately describes transmission coefficients measured up to 2.9 J cm-2.

Changes in spectral shape of tissue optical properties in conjunction with laser-induced thermotherapy.

We measured the optical properties on samples of rat liver tissue before and after laser-induced thermotherapy performed in vivo with Nd:YAG laser irradiation. This made it possible to monitor not only the influence of coagulation on the scattering properties but also the influence of damages to vessels and heat-induced damage to blood on the absorption properties. An experimental integrating-sphere arrangement was modified to allow the determination of the g factor and the absorption and scattering coefficients versus the wavelength in the 600-1050-nm spectral region, with the use of a spectrometer and a CCD camera. The results show a relative decrease in the g factor of on average 21 ? 7% over the entire spectral range following thermotherapy, and a corresponding relative increase in the scattering and absorption coefficients of 23 ? 8% and 200 ? 100%, respectively. An increase of on average 200 ? 80% was consequently found for the reduced scattering coefficient. The cause of these changes in terms of the Mie-equivalent average radius of tissue scatterers as well as of the distribution and biochemistry of tissue absorbers was analyzed, utilizing the information yielded by the g factor and the spectral shapes of the reduced scattering and absorption coefficients. These results were correlated with the alterations in the ultrastructure found in the histological evaluation. The average radius of tissue scattering centers, determined by using either the g factors calculated on the basis of Mie theory or the spectral shape of reduced scattering coefficients calculated on the Mie theory, was estimated to be 21-32% lower in treated than in untreated liver samples. The Mie-equivalent average radii of scattering centers in untreated liver tissue deduced by the two methods corresponded well and were found to be 0.31 and 0.29 mum, respectively, yielding particle sizes in the same range as the size of a mitochondrion.

T-matrix computations of light scattering by red blood cells.

The electromagnetic far field, as well as the near field, originating from light interaction with a red blood cell (RBC)volume-equivalent spheroid, was analyzed by utilizing theT-matrix theory. This method is a powerful tool thatmakes it possible to study the influence of cell shape on the angulardistribution of scattered light. General observations were that thethree-dimensional shape, as well as the optical thickness apparent tothe incident field, affects the forward scattering. Thebackscattering was influenced by the shape of the surface facing theincident beam. Furthermore sphering as well as elongation of anoblate RBC into a volume-equivalent sphere or a prolate spheroid, respectively, was theoretically modeled to imitate physiologicalphenomena caused, e.g., by heat or the increased shear stress offlowing blood. Both sphering and elongation were shown to decreasethe intensity of the forward-directed scattering, thus yielding lowerg factors. The sphering made the scattering patternindependent of azimuthal scattering angle phi(s), whereas the elongation induced more apparent phi(s)-dependent patterns. The lightscattering by a RBC volume-equivalent spheroid was thus found to behighly influenced by the shape of the scattering object. Anear-field radius r(nf) was evaluated as thedistance to which the maximum intensity of the total near field haddecreased to 2.5 times that of the incident field. It was estimatedto 2-24.5 times the maximum radius of the scattering spheroid, corresponding to 12-69 mum. Because the near-field radiuswas shown to be larger than a simple estimation of the distance betweenthe RBC's in whole blood, the assumption of independent scattering, frequently employed in optical measurements on whole blood, seemsinappropriate. This also indicates that one cannot extrapolate theresults obtained from diluted blood to whole blood by multiplying witha simple concentration factor.

Near infrared diffuse reflection and laser-induced fluorescence spectroscopy for myocardial tissue characterisation.

In order to evaluate the potential of cardiovascular tissue characterisation using near-infrared (NIR) spectroscopy, spectra in a previously unexplored wavelength region 0.8-2.3 micron were recorded from various pig heart tissue samples in vitro: normal myocardium (with and without endo/epicardium), aorta, fatty and fibrous heart tissue. The spectra were analysed with principal component analysis (PCA), revealing several spectroscopically characteristic features enabling tissue classification. Several of the identified spectral features could be attributed to specific tissue constituents by comparing the tissue signals with spectra obtained from water, elastin, collagen and cholesterol as well as with published data. The results obtained with the NIR spectroscopy technique in terms of its potential to classify different tissue types were compared with those from laser-induced fluorescence (LIF) using 337 nm excitation. LIF and NIR spectroscopy can in combination with PCA be used to discriminate between all previously mentioned tissue groups, apart from fatty versus fibrous tissue (LIF) and aorta versus fibrous tissue (NIR), respectively. The NIR analysis was improved by focusing the PCA to the wavelength segment 2.0-2.3 microns, resulting in successful spectral characterisation of all cardiovascular tissue groups.

Changes in optical properties of human whole blood in vitro due to slow heating.

Optical properties of human whole blood were investigated in vitro at 633 nm using a double integrating sphere set-up. The blood flow was maintained at a constant rate through a flow cell while continuously heating the blood at 0.2-1.1 degrees C/min from approximately 25 to 55 degrees C in a heat exchanger. A small, but rather abrupt decrease in the scattering asymmetry factor (g-factor) of 1.7 +/- 0.6% and a similar increase in the scattering coefficient of 2.9 +/- 0.6% were observed at approximately 45-46 degrees C yielding an increase in the reduced scattering coefficient of 40 +/- 10%. Furthermore, a continuous, manifest increase in the absorption coefficient was seen with increasing temperature, on average 80 +/- 70% from 25 to 50 degrees C. The effect of the heating on the blood cells was also studied under a white-light transmission microscope. A sudden change in the shape of the red blood cells, from discshaped to spherical, was observed at approximately the same temperature at which the distinct changes in g-factor and scattering coefficient were observed, i.e. at 45-46 degrees C. The results indicate that this shape transformation could explain the sudden change in scattering properties.

Detection of adenocarcinoma in Barrett's oesophagus by means of laser induced fluorescence.

PATIENTS: Seven patients with Barrett's metaplastic epithelium and oesophageal adenocarcinoma were investigated by means of laser induced fluorescence after low dose intravenous injection (0.35 mg/kg bw) of Photofrin (QLT, Vancouver, Canada). Laser induced fluorescence measurements were performed immediately after resection of the oesophagus. METHODS: Laser induced fluorescence spectra were recorded from 15-30 locations in each surgical specimen from normal mucosa, Barrett's epithelium, and tumour tissue. Histological examination was performed on each location to correlate the fluorescence spectral characteristics with histological status of the epithelium (normal, metaplastic or malignant). Measurements were also performed during endoscopy in five patients to test the applicability of the method in a clinical setting. Fluorescence spectra were recorded and evaluated at characteristic wavelengths, and biopsy specimens were collected. Fluorescence ratios were calculated as the quotient of Photofrin fluorescence divided by autofluorescence. RESULTS: The mean (SD) fluorescence ratio values were 0.10 (0.058) for normal oesophageal mucosa, 0.16 (0.073) for normal gastric mucosa, 0.205 (0.17) for Barrett's epithelium with moderate dysplasia, 0.79 (0.54) for severe dysplasia, and 0.78 (0.56) for adenocarcinoma. The highest fluorescence ratios were obtained for adenocarcinoma tissue, which could generally be distinguished from all nonmalignant tissue. Metaplastic Barrett's epithelium also yielded higher fluorescence ratios than did normal mucosa. CONCLUSIONS: The results suggest that the technique can be used during endoscopy for real time tissue characterisation in the oesophagus, as an aid in detecting malignant transformation not macroscopically apparent at endoscopy.

Pro- and antifibrinolytic effects of ultrasound on streptokinase-induced thrombolysis.

We have earlier documented that ultrasound can enhance the fibrinolytic effect of streptokinase and shorten the reperfusion time under experimental conditions. The present study concerns the magnitude of the effect in relation to ultrasound frequency, intensity and exposure time. The effect of ultrasound on the structural properties of the fibrin network was evaluated by scanning electron microscopy. Whole blood clots were created and exposed to streptokinase (5000 U/mL). In paired experiments, one clot was also exposed to pulsed ultrasound. Lysis is expressed as the percentage decrease in clot weight. Clot lysis was equally affected by ultrasound in the range of 0.5 to 2.3 MHz. Below 0.5 W/cm2 no enhancing effect was observed, while intensities between 0.5 and 1.5 W/cm2 resulted in a significant enhancement of clot lysis. Intensities above 4 W/cm2 inhibited the fibrinolytic effect of streptokinase but not of rt-PA. From scanning electron microscopy there was no evidence that ultrasound disrupted the clot surface or damaged the fibrin structure.

Measurements of the optical properties of tissue in conjunction with photodynamic therapy.

A simple optical dosimeter was used to measure the light intensity in rat liver and muscle in vivo with fibers positioned at different depths to investigate whether the light penetration changed during photodynamic therapy (PDT). The results were then correlated with measurements of the three optical-interaction coefficients µ(s), µ(a) and g for wavelengths in the range 500-800 nm for PDT-treated and nontreated rat liver and muscle tissue in vitro. Adistinct increase in the absorption coefficient was seen immediately after treatment, in agreement with the decreasing light intensity observed during the treatment, as measured with the optical dosimeter. The collimated transmittance was measured with a narrow-beam setup, and an optical integrating sphere was used to measure the diffuse reflectance and total transmittance of the samples. The corresponding optical properties were obtained by spline interpolation of Monte Carlo-simulated data. To ensure that the measured values were correct, we performed calibration easurements with suspensions of polystyrene microspheres and ink.

Enhancement of thrombolysis by ultrasound.

Drug-induced early reperfusion in acute myocardial infarction reduces myocardial damage and decreases mortality. A further beneficial effect may be achieved if the time from start of thrombolytic treatment to reperfusion, on average 45 min, can be shortened. With this purpose in mind, we have analysed the effect of ultrasound on the reperfusion time in an experimental model in vitro. A cylindrical fibrin thrombus with a 2 mm diameter and a 20 microL volume was made by thrombin activation of a pure 0.5% fibrinogen solution in a soft silicone tube. The tube was placed in a low pressure perfusion system and maintained at 37 degrees C. The thrombi were then exposed to hydrostatic loading with a streptokinase concentration of 5000 units/mL. Reperfusion times (RT) were measured from time of Streptokinase exposure to fluid passage, identified by the photoelectric technique. RT increased significantly with increasing thrombus age (r = 0.92, p < 0.05) and was 34-45 min (95% confidence limits) at a thrombus age of 1 h and 102-122 min at a thrombus age of 2 h. RT was unaffected by temperatures between 33 and 45 degrees C but increased with higher temperatures. All investigations of ultrasound effects were performed with 1 h old thrombi and at 37 degrees C. RT decreased by 49% (p < 0.01) as an effect of exposure to 1 MHz ultrasound at 1 W/cm2 SATA. Intermittent ultrasound exposure for 10 microseconds/ms with the same intensity and frequency shortened RT by 54% (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption, retention and excretion of 65Zn in rats with wounds treated with 65Zn-labelled zinc tape.

Absorption, retention and excretion of 65Zn in rats from excisional wounds treated with 65Zn-labelled zinc tape were studied. The absorption of 65Zn was marked and indicates that the absorption of zinc from the tape is more than sufficient to account for the increases in zinc concentrations seen in several tissues. The distribution, retention and excretion of 65Zn from the zinc tape followed a pattern similar to that of 65Zn injected into non-operated and operated rats. This indicates that the zinc from the tape is present in the body not as zinc oxide, as it is in the adhesive substance of the tape, but as zinc in those forms which are originally found in the body and that zinc oxide is ionized before absorption. In tissues with a rapid uptake of zinc more than 50% of the total concentration of zinc derived from the zinc tape after eight days of treatment. In operated animals which were not treated with zinc tape a decline was seen in the serum zinc concentration and excretion of zinc with a concomitant increase in the liver concentration of zinc.